1

Journal of Phycology Achimer February 2017, Volume 53, Issue 1, Pages 161-187 http://dx.doi.org/10.1111/jpy.12489 http://archimer.ifremer.fr http://archimer.ifremer.fr/doc/00356/46718/ © 2016 Phycological Society of America

Morphological and genetic diversity of Beaufort Sea with high contributions from the neogracilis species complex

Balzano Sergio 1, *, Percopo Isabella 2, Siano Raffaele 3, Gourvil Priscillia 4, Chanoine Mélanie 4, Dominique Marie 4, Vaulot Daniel 4, Sarno Diana 5

1 Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR7144, Station Biologique De Roscoff; 29680 Roscoff, France 2 Integrative Marine Ecology Department, Stazione Zoologica Anton Dohrn; Villa Comunale 80121 Naples ,Italy 3 IFREMER, Dyneco Pelagos; Bp 70 29280 Plouzane ,France 4 Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR7144, Station Biologique de Roscoff; 29680 Roscoff ,France 5 Integrative Marine Ecology Department; Stazione Zoologica Anton Dohrn; Villa Comunale 80121 Naples, Italy

* Corresponding author : Sergio Balzano, email address : [email protected]

Abstract :

Seventy-five diatoms strains isolated from the Beaufort Sea (Canadian Arctic) in the summer of 2009 were characterized by light and electron microscopy (SEM and TEM) as well as 18S and 28S rRNA gene sequencing. These strains group into 20 genotypes and 17 morphotypes and are affiliated with the genera Arcocellulus, , Chaetoceros, Cylindrotheca, , Nitzschia, Porosira, Pseudo- nitzschia, Shionodiscus, , Synedropsis. Most of the species have a distribution confined to the northern/polar area. Chaetoceros neogracilis and Chaetoceros gelidus were the most represented taxa. Strains of C. neogracilis were morphologically similar and shared identical 18S rRNA gene sequences, but belonged to four distinct genetic clades based on 28S rRNA, ITS-1 and ITS-2 phylogenies. Secondary structure prediction revealed that these four clades differ in hemi-compensatory base changes (HCBCs) in paired positions of the ITS-2, suggesting their inability to interbreed. Reproductively isolated C. neogracilis genotypes can thus co-occur in summer communities in the Beaufort Sea. Chaetoceros neogracilis generally occurred as single cells but can also form short colonies. It is phylogenetically distinct from an Antarctic species, erroneously identified in some previous studies as C. neogracilis but named here as Chaetoceros sp. This work provides taxonomically validated sequences for 20 Arctic taxa, which will facilitate future metabarcoding studies on phytoplankton in this region.

Keywords : biogeography, ITS, ITS2 secondary structure, LSU, morphology, phylogeny, polar diatoms, SSU

Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site. species belonging to the generaspecies tothe belonging and Lovejoy & 1997, Horner etal.2002) (Booth phytoplankton, locations in coastal especially Wang etal.2005). et al.2003, production (Sherr daylightand September,leadto March between inabundance variability undergo seasonal high INTRODUCTION Accepted during upwelling Except episodic events, (Pickart etal.2013). growth promote phytoplankton and layer uptothesurface nutrient richwaters canbring periodic wind-drivenupwelling events primary productionandphytoplankton blooms playsMackenzie River, which roleindisrupting akey the wintericeinearlyspring promoting Ratkova & 2002). Wassmann etal.2002, Booth phytoplankton(Tuschling etal.2000, communitiesindifferent regions A Due tofluctuationsinlight,temperature, salini The Beaufort Sea is a major component of the Arctic Ocean, and is highly influenced by the by and is highly Ocean, influenced component of theArctic Seaisamajor The Beaufort This article is protected by copyright. All rights reserved. AllrightsThis articleis protectedby reserved. copyright. Chaetoceros Ehrenberg and Ehrenberg (Carmack & MacDonald 2002). In& addition, MacDonald 2002). (Carmack and composition. Higher temperatures and longer composition.Higher temperatures and an increase in algal biomass andprimary inalgal biomass increase an Diatoms account for a high portion of Arctic portionofArctic a high Diatoms account for ty and sea ice extent, Arctic phytoplankton Arctic tyextent, andseaice Thalassiosira Cleve candominate on photosynthetic populations (Balzano et al. 2012b) partially agree with pigment analyses and analyses withpigment agree partially on photosynthetic(Balzano etal.2012b) populations [cloning/sequencingand ter Molecular techniques nordenskioeldii, 2013), several other Acceptedsocialis of ecotype diatoms etal.2015).Within thecold-water Shelf(Coupel Mackenzie whilediatomsacc waters dominated offshore Dinophyceae and Mamiellophyceae thatPrymnesiophyceae, composition andrevealed (http://malina.obs-vlfr.fr/data.html) confirmed techniques andlight microscopy (Coupel etal.2015) mid-August. analyses Pigment multidisciplinary until anextensive undertaken was to theBeaufort effort sampling Seawhere Article (Brugelcommunity etal.2009). withdiatoms along Autumn communitiesincludehigher contributions candominatethe ofdinoflagellates, which Sukhanova etal.2009). (DCM; thedeep chlorophyll maximum bloom morefrequently at (Hill etal.2005)anddiatoms community insummer diatoms tothephytoplankton increase spring etal.2005,Sukhanova2009) (Hill blooming inlate etal.2005),occasionally (Hill nearthecoast abundant tendtobemore Diatoms Micromonas Micromonas psychrophilicmostly byrepresented the ofpicoeukaryotes, low, leading totheprevalence nutrien column ishighlystratified, the the water The MALINA oceanographic expedition sailed in July 2009 from the Pacific coast of Canada coastofCanada sailedin expedition The MALINAJuly oceanographic 2009fromthePacific described recently as described recently This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. ”

Manton & Parke ecotype corresponding to the single genetic “Arctic clade tothesingle named corresponding ParkeManton & ecotype (Lovejoy et al. 2007, Balzano et al. 2012b), withinthephytoplankton community.2012b), al.2007,Balzano etal. et (Lovejoy and Chaetoceros Pseudo-nitzschia Pseudo-nitzschia Chaetoceros gelidus gelidus Chaetoceros taxa, and with lower abundances, andwithlower abundances, taxa, spp. prevailed (http://malina.obs-vlfr.fr/data.html). spp. prevailed ounted for most abundance and biomass onthe biomass and abundance formost ounted previous findings previous community onphytoplankton . The algal biomass and the contribution of algal biomassandthe The . t concentration in the upper layers isextremely t concentrationintheupper minal-RFLP the18SrRNAgene] on (T-RFLP) (Degerlund et al. 2012, Chamnansinp etal. (Degerlund etal.2012, Thalassiosira Thalassiosira Chaetoceros phytoplankton microscopycount during the MALINA cruise. Photosynthetic pico- and nanoeukaryotic populations were populations and nanoeukaryotic Photosynthetic pico- cruise. during theMALINA tophotosynthetic collected applied been approachhas species identification.This % of the99.5 toseparate 2) hasbeenproved socialis (2- the only 2 (< picoplankter photosynthetic fragment extending from the5 fragmentfrom extending gene A (Lee etal.2013). species discriminatorycentric diatom among marker molecular et al.2010) withinthegenera mostofthespecies gene successfully discriminate can resolution power:the28SrRNA ahigher ribosomalgeneshave al. 2012b).Other (Balzano et AcceptedChaetoceros Similarly,gene didnotallowdisc the18SrRNA fine ultrastructuralthe observation detail ofthe Articlemethods employed. microscopy,Light hasbeen that tothelimited resolution due power and molecular assessed for of diatoms, themorphological Beaufort Sea,butsp the partiallyelucidated for 2012b). 20 Coupling cultureisolationwithmorphologica Seasonal succession and geographic distribution and Seasonal succession μ m) geneticlibrari )

and This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright.

and and Chaetoceros neogracilis Chaetoceros Pseudo-nitzschia Pseudo-nitzschia es were dominated by the diatoms dominatedbyes were the ’ end ofthe5.8Sto3’e s indicating that Arctic s indicating that (Lundholm 2002)andisconsideredagood etal. H.Peragallo, intheareawhich were well represented in DCM and surface waters, respectively (Balzano etal. (Balzano respectively in DCMandsurfacewaters, μ m) detected inmoststations,whereas m) detected nanoplankton diatom species (Moniz & Kaczmarska 2010). 2010). &(Moniz Kaczmarska species diatom s oftenrequired diatomspecies. todistinguish ecies level diversity hasstillnotbeenfully leveldiversity ecies l and genetic characterization allowsdetailed genetic characterization l and rimination among some species of the genera genera ofthe among somespecies rimination of phytoplankton species have thus been of phytoplanktonhave thusbeen species applied in most thestudies,doesnotallow appliedinmost Micromonas Micromonas nd of the helix IIIITS of helix nd ofthe C. gelidus (Lovejoy et al.2007) (Lovejoy (referred thereinas Chaetoceros -2 (5.8S + ITS--2 (5.8S+ (Kooistra C.

was strains, 60ofwhich are (March2016) currently Gall et al.2008,Balza (Le described previously Phytoplankton strainswere isolatedbothonboard withNiskinbottl depths Pacific and atdifferent w collected SupportingInformation). Sampleswere 1/08/09to24/08/09(Table outinlatesummer from carried sampling S1inthe was effort 06/07/09 from Victoria(BritishCanada Columbia, whichsailedthe cruise samples collectedthe MALINA during (http://www.obs-vlfr.fr/Malina) MATERIALS METHODS AND reproductive isolation. th of secondary structure the and we reconstructed to and 28SrRNAgenesequences Accepted Articlea numberof gene from rRNA ITS ofthe operon toidenti with 18Sand28SrRNAgene sequencing isolatedfromthe we focusondiatomstrains butsimilarinformati et al.2013), (Guapproaches 2012a). light by microscopy (LM) both and characterized were andHeterokontophyta isolated Chlorophyta, divisions Haptophyta, Cryptophyta Dinophyta, belonging al.2012b)and104strains tothe et (Balzano dominated bycultured microorganisms Phytoplankton sampling,isolationandmaintenance A recent study investigated Arcticdinofl studyA recent investigated This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. further investigate theoccurren investigate further agellates couplingand genetic agellates morphological Beaufort Sea. We combined LM, TEM,andSEM combined Sea.We Beaufort es intheBeaufort Sea. mounted onaCTDframe no et al. 2012a). Overall we isolated 75diatom we Overall no etal.2012a). available from the Roscoff Culture Collection fromtheRoscoff available C. neogracilis C. on diatomsismissing.In thepresent paper, and 18S rRNA gene sequencing (Balzano etal. (Balzano genesequencing and 18SrRNA and back in the laboratory (Table 1) as 1) (Table and backinthelaboratory e ITS-2 of these strainsinordertopredict their ofthese ITS-2 e fy the isolated strains. We also sequenced the fy strains. alsosequenced We the isolated ) to the Beaufort Sea where an extensive extensive an Seawhere ) totheBeaufort ith a bucket from surface waters intheNorth waters ith abucket from surface . Strains were isolated from seawater fromseawater isolated . Strainswere strains ce of distinct genetic entities ce ofdistinct

sharing similar18S highly of 35 and kept at 4°C at an irradianceof50 of 35andkeptat4°Can medium (Kelleral. 2009)withadditionofsilicate, etfrom seawater prepared sterile at asalinity comparison purposes for cruise of theMALINA duringfirst leg the sampled theNorthPacific includedfourstrains from also Beaufort Seabutwe (RCC: http://www.roscoff-culture-c m Some of the neogracilis belongand Microbiota (Bigelow, thoughtfor to USA)andpreviously Algae Marine to neogracilis 3’) were used 63f (5’ by PCRongenom thenamplified rRNA genewere Hoerdt, France) andfollowing theinstructions by provided themanufacturer. etal.2012a)using (Balzano described previously the genus μ M) to induce resting spore formation, since spor M) toinduce formation,since resting spore Accepted Article . -2 The ITS of rRNA operonwasamplified fromThe region 35MALINA ofthe strains and the28S operon oftherRNA (ITS) Spacer Internalgene, Transcribed The 18SrRNA the DNA extractionandPCR. s -1 -ACGCTT-GTC-TCA-AAG-ATTA- ) inf/2medium(Guillard 1975)withnitra Chaetoceros This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. (CCMP187, CCMP189, andCCMP190; TableS2intheSupportingInformation). 3Antarctic strainsof and (Table 1) C. neogracilis (Lepère et al. 2011) as described previously (Balzano et al. 2012a). etal.2012a). (Balzano previously al. 2011)asdescribed (Lepère et (Hasle & Syvertsen 1997). 1997). & (Hasle Syvertsen strains were incubated at lowlight strainswere 10 intensity incubated (about Genomic DNA was extracted from 75 MALINA strains from75MALINA Genomic as DNAwasextracted ollection.org/). Most of the strains were isolated fromthe Mostofthestrainswere ollection.org/). μ 3’) and 1818r(5’ and 3’) mol photons Chaetoceros Chaetoceros . The strains were maintained in K or K/2- in maintained . Thestrainswere e morphology can help species identificationin can helpspecies e morphology te supplied at a concentration 10-fold lower(88 te suppliedataconcentration 10-fold the NucleoSpinTissuekit(Mackerey Nagel, ic DNA. For the 18S rRNA gene the primers genetheprimers the18SrRNA ic DNA.For . m purchased from theNationalfrom Centre purchased -ACG-GAAACC-TTG-TTA-CGA- -2 . -2 s -1 ina12:12 light darkregime. μ mol photons C. C.

. based on 99.5% sequences similarity, Bioe usingbased on99.5%sequences the (http://www.ebi.ac.uk/Tools/msa/clustalw2) and ClustalW2 using BLAST aligned (blast.ncbi.nlm.nih.gov/Blast.cgi), wasABI carried3100 sequencer(AppliedBiosystems). outonan prism primer D1R wasus forward above whereas the ITSregion using primers described selected wassequenced and reverse bothforward strains. The 63fand1818Rwere theprimers used 2005), whereas (Zhu etal. Euk528f from primer theinternal allthestrainsusing sequenced rRNA genewas al. 2012)of the18S et (Dunthorn V4region USA). Thehypervariable city, Biosystems, Foster deter were sequences Clara, USA)andpartial cycles 1min,55°Cformin30s,and72°Cmin. of94°Cfor Orsinietal.2002). et al.1989, rRNA (Lenaers ACG-AAC-GAT-TTG-CAC-GTC-AG- was amplified using D1R(5’ primers elongationstepat72°Cfor7 15 s)and afinal for1min 95°C during for1min,55°C45s,and72°C 5min,35amplification cycles (95°C includedan respectively. initial etD1R (Lenaers PCRcondition al.1989), incubation stepat etal.2004)andthe28Sforwardprimer (Guillou 18S 329r primerfor complements ofthereverse Accepted ArticleD1R- ITS (5’ primers329f The wasamplifiedusing Phylogenetic analysis. ITS,18S rRNA, 28S rRNAampliconswere and R (5’ - TAT-GCT-TAA-ATT-CAG-CGG-GT- This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. V4 sequences were compared to those available in Genbank using in were tothoseavailable V4 sequences compared -ACC-CGC-TGA-ATT-TAA-GCA-TA- 3’) targeting3’) theD1– mined bymined using Big TerminatorV3.1(Applied Dye -GTG-AAC-CTG-CRG-AAG-GAT-CA- min. From 72 diatom strains, the28SrRNAgene min. From 72diatomstrains, ed to sequence the 28S rRNA gene. Sequencing gene. Sequencing to sequencethe28SrRNA ed PCR reactions were as follows: 30amplification asfollows: PCR reactionswere then grouped into 17 different 18Sgenotypes then groupedinto17different purified using Exosap (USB products, Santa products, (USB using Exosap purified 3’) which correspond to the reverse thereverse to correspond 3’) which dit software (Hall 1999). Thefull18S rRNA 1999). (Hall dit software to sequence the full 18S rRNAgene from thefull18S tosequence D3 region of the nuclear LSU nuclear D3 region ofthe 3’) and D3Ca(5’ 3’) 3’) and - C. neogracilis the genus from sequences & and Nei (Tamura 1993) model aTamura-Nei phylogeny using inferred was andthe positions and490nucleotide included35sequences pennate diatoms,thealignment AcceptedAttheya Phylogenetic were as then inferred relationships 1980). model(Kimura Kimura-2 504 positionsandthephylogeny a inferred using was and thealignment65sequences included, centric diatoms, Forthe removedalignments. fromthe and trees (centricdiatoms,pennatediatoms genotyp onesequenceper at least subset, containing andMLas thebestmodeltoinfer bothNJ 18Sphylogeny. Article positions.ATamura contained 1,465nucleotide regionsHighly ofth variable above. as described usingclustalW2 (http://www.ncbi.nlm.nih.gov/nucleotide, for atotalof84sequences S2) Table theML topology. treesbased on thephylogenetic toconstruct et al.2011)was used (Felsenstein 1985) estimated using 1,000replicates valueswere & 2000)andbootstrap (Nei Kumar (ML) methods and Joining Neighbour (NJ) Likelihood usingMaximum analyzed were inthispaper,relationships treesshown phylogenetic all the For (19strains intotal). genotype atleastgene from onestrainper was sequenced For the D1-D3 region of the 28S rRNA gene 64 sequences were aligned usingand a werethe 28SrRNAgene region clustalw2 64sequences of For theD1-D3 aligne Full were 18SrRNAsequences West were used as an outgroup and were then andwere as anoutgroup wereWest used Attheya This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. , whichincluded36MALINA fromth strains were also used as an outgroup. A third phylogenetic tree was constructed for was A thirdphylogenetictree constructed usedasanoutgroup. also were d with reference sequences from Genbank from d withreference sequences C. neogracilis C. neogracilis described above and 5 sequences from the genus the from above and5sequences described Nei model (Tamura & Nei 1993) was selected 1993)wasNei selected & (Tamura Nei model e alignment were removed and the final dataset andthefinaldataset removed were e alignment for both methods. MEGA5 software (Tamura software for bothmethods.MEGA5 e, was used to construct three phylogenetic was threephylogenetic usedtoconstruct e, removed from the tree for clarity. For the For clarity. removed fromthetreefor strains). Highly variable regionsstrains). Highly were is species, 1 sequence of the strain ofthe is species,1sequence based on the secondary structure of the ITS-2 from ITS-2 from ofthe based onthesecondary structure &III alignment Cantor1969).Formodel (Jukes theend the5.8/ITS-2 ofhelix annotated was 227nucleotidepositions and included 30sequences ITS-1 alignment orthe5.8S/ITS-2. The ITS-1 the fromthealignment ofeither wereexcluded ITS-2. Someseque conservedIII motifofhelix of consisting inaregion tree starting atthe another phylogenetic Wethenconstructed 2010) availableinGenBank. from other 1 and5.8Sbasedonsequences atinterspecif 5.8S isaregion conserved highly C. neogracilis model. positionsusingfor atotalof590 aKimura-2-parameter sequences gelidus Antarctic strainsCCMP163,CCMP189andCC AcceptedITS-1 alignments, joininglong and the5.8S/ITS-2 aneighbour phylogenetic tobeincludedinthe RCC2268,RCC2277 andRCC2318 from ITS-2were1 andthe5.8+ sequences notsufficiently IIE43.N2, butitwasattributedtoClade based strainMALINA fromITS the not besequenced could 1980).The a Kimura-2 model(Kimura positionsandboth and 384nucleotide 30 sequences alignment included final reconstructed. The ITS-2 hasbeen ofthe structure which thesecondary genus closelyrelatedwhich isthespecies tothe (Sorhannus most etal.2010), Article CPH9 identifiedas We also sequenced the ITS operonoftherRNA We alsosequencedthe (RCC2271) which (RCC2271) This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. as well as the Antarctic strains attributed by CCMP to attributedbyas welltheAntarctic strains CCMP Chaetoceros fallax

was used as an outgroup. The analysis was performed on41 was Theanalysis performed wasan outgroup. usedas Prosckina Lavrenko, 3 GenBank sequences from the from Lavrenko, Prosckina sequences 3GenBank ic level, we identified the boundary between ITS- between boundary weidentifiedthe ic level, on its 28S sequence. Similarly since both the ITS- its28S sequence.Similarly boththe since on Chaetoceros Chaetoceros MP190 (Table S2) and one sequence from sequence MP190 (TableS2)andone nces did not cover the entire ITS and length entire nces didnotcover the a phylogenetic tree based on the ITS-1 and ITS-1 onthe tree based a phylogenetic gene from the MALINA strains affiliatedto strains gene fromtheMALINA T. weissflogii and was analysed using a Jukes Cantor usingaJukes andwasanalysed ML and NJ phylogenies were inferred using using inferred were ML phylogenies andNJ species (Moniz & (Moniz Kaczmarska species 5’ end of 5.8S andending end inthe of5.8S 5’ (Grunow)Fryxell& Hasle C. neogracilis Chaetoceros . Since the C. for

strains. http://www.ros at available areUSA). Micrographs imagedwith aSPOTRT-sliderdigitalInstruments,(Diagnostics camera Sterling Heights, MI, usingwith a100Xobjective Germany) differen Hamburg, BX51 observed microscope (Olympus, using anOlympus growth and phase oftheir inlight microscopy. observed andphotographed etal.2012). (Merget database other transferred onto etal.2004)andthen (Mathews RCC2014 using the RNAstructureprogram for thestrain wasITS-2 firstinferred ofthe regions Thesecondarystructure et (Keller al.2009). Modelsoftheflanking 5.8Sand28S usingannotated boundaries wereHiddenMarkov then ITS-2 The therRNA. operonof ITS-2 the secondary of structure detail wereconstructed the strains. genetic these toidentifythe SupportingInformation) theclade was inorder of constructed positions(Fig.for atotal483 S1in sequences ITS 34 fragment entire whichincluded tree forthe Accepted 1).To Stazione Zoologica Dohrn(Table Anton Light (28strains)observed (25strains) at using Microscopy and/orSEM TEM (36strains), Article Selected strains, covering most geneticSelected strains, most covering di Microscopy ITS-2 structureprediction. Chaetoceros This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. . At least one strain per genotype, for a total of 61 strains (Table 1),was atotalof61strains(Table for genotype, one strainper . Atleast sequences through homology modelling (Wolf et al. 2005) using the ITS-2 using the through homology(Wolf etal.2005) modelling sequences To characterize our MALINA strainsof our To characterize versity based on both 18S and 28S rRNA,werebased onboth18Sand28S versity tial interference contrast (DIC). Cellswere interference tial remove organic matter, organic matter, remove Cells were harvested during the exponential wereexponential harvested duringCells the coff-culture-collection.org for a large set of for setof alarge coff-culture-collection.org samples were treated were treated samples C. neogracilis C. neogracilis in deeper with nitricand sulfuric acids(1:1:4,sample:HNO Accepted generawere 3).Themost represented and (Figs.genetic 2 clades that 36strains of (Figs.based on18Sand28SrRNAphylogenies affiliatedto the rRNA fromallthestrains ITS operonof and the from mostofourstrains the28SrRNA wesequenced genotype. Moreover each unique atleastonestrainfrom thefull18SrRNAfrom strains andthenwe sequenced sequence (Table1).We and moleculartechniques RESULTS Article withSEM. and observed sputter-coated filters were mountedonstubs, Dried 100%),andseries (25,50,75,95,and critical-point-dried. an ethanol Pleasanton, withdistilledwater, CA,USA)filters, rinsed polycarbonate in dehydrated onNuclepore 3 samplesnotsubjectedtocleaningFixed wereplaced Inc., USA). Peabody, SEM(JEOL-USA MA, with aJEOLpalladium andobserved JSM-6500F and/or Germany) Oberkochen, microscope (LEO, LEOgridswith a electron 912AB andobserved mounted onFormvar- transmission coated Zeiss, Obe Digitala Axiocam (Carl Camera LMwith distilledwater.observations perf were In characterized the presentstudy, we 75diatomstrainsa combinationofmorphological using

This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. C. neogracilis sharing identical 18S rRNA gene sequence makesharing up4distinct 18SrRNAgene identical sequence C. neogracilis. C. neogracilis. rkochen, Germany). Acid-cleaned material was materialwas Acid-cleaned Germany). rkochen, ormed using a Zeiss Axiophot using 200equippedwith ormed a 1 and 2). 28S rRNA and ITS analyses ITS indicate and 2).28SrRNA 1 3 mounted on stubs, sputter-coated withgold- mounted onstubs,sputter-coated :H d theV4region rRNAfromallthe ofthe18S 2 SO 4 The strains grouped into17genotypesThe strainsgrouped ), boiled for a few seconds and washed secondsandwashed ), boiledfor afew Chaetoceros μ m pore size (Nuclepore, m poresize and Thalassiosira . Nitzschia Cylindrotheca branches with two other sequences from sequences with twoother branches from et al.2007).The28SrRNAgene sequence lineag ofthetwo cluster toany clad forming amoderately-supported sequences Kusber 2005). & Hasle1964,Jahn 4A; (Fig. to thevalveface and joineddirectly are irregularly narrow, spaced, (13-17in10 Thefibulae nodule. a central rapheisinterruptedby silicified ribs.Thecentral slightly bytransapical transversed face isunperforated, & Thevalve 1964, Jahn Kusber2005). nitzschia sequence from It obser beenrepeatedly al. 2007). has (Haitao et to

Accepted Article constitute a species complex ofsimilarmorphotyp complex constitute aspecies Bacillariaceae Cylindrotheca closterium Cells are 85to108 Cylindrotheca closterium species. and This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. Pseudo-nitzschia. and C. closterium . Nitzschia We isolated 9 Bacillariaceae strains from the genera fromthegenera strains isolated9Bacillariaceae We μ m long, fusiform with rostrated ends and possess two chloroplasts (Hasle endsandpossesstwochloroplasts m long, withrostrated fusiform was considered as a cosmopolitan species but it was demonstrated it was demonstrated speciesa cosmopolitan but as considered was (Ehrenberg) Lewin & Reimann. Lewin & (Ehrenberg) Reimann. representatives but was poorly resolutive for the different different poorlyresolutive butwas forthe representatives strain RCC1985 (Fig. 1) groups with the other groups strainRCC1985(Fig. 1) withthe es described to date for the todate es described The 18S rRNA gene (Fig. 1) discriminated the different thedifferent 1) discriminated gene (Fig. The 18SrRNA C. closterium. C. closterium. e (sequence similarity > 97.8 %),butdoesnot > similarity (sequence e ved in the Arctic (Table 2).The 18S rRNA gene gene 18SrRNA (Table 2).The intheArctic ved C. closterium es belonging to different genetic lineages genetic lineages todifferent es belonging C. closterium strain RCC1985(Fig. 2A) species complex (Haitao (Haitao complex species Cylindrotheca, C. closterium Pseudo- μ m) of the valves have striae barelyof thevalves striae silicifiedth have μ in10 The striae (54-55 rounded. 4E). Apicesare (Fig. theotherconvex of thevalvesand is linear swelling, withacentral were lanceolate conditions.Valves oneside are notobserved inculture Nitzschia genus the describing (2002) Lundholmspeciessupports theassertionof etal. (Fig. 2A),which with different branches Thisclade 97.5 and97.4,respectively). strain RCC2276groupswith branches with other relatedtothatof strain RCC2276ishighly (Table environments European freshwater interspace is present (Fig. 4, C and D). 4,CandD). (Fig. interspace ispresent in 10 Thedens ingirdle view. whenrectangular observed (Fig. 4B),and thepoles,invalveview towards tapering chloroplasts. Cellsare lanceolate,

AcceptedIn RCC2006,most thestrain in5-7sectors. ofporoidsdivided am) are row single composedof Article Cells (apical 17-25 axis: Pseudo-nitzschia granii Cells (apical 35 axis: Nitzschia pellucida Nitzschia pellucida μ m, respectively. Each stria contains on m, respectively. striacontains Each as polyphyletic. This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. Nitzschia has been previously reported in Arctic has been previously in reported Grunow. μ m; 3.0-3.5 transapical axis: (Hasle) Hasle μ Nitzschia laevis m; transapical axis: 1.4-1.8 1.4-1.8 m; transapicalaxis: species 28SrRNAgene (Fig.from 1).The sequence . . 2). The 18S rRNA gene sequence from 18SrRNAgene sequence 2). The at lack complete poroidsat lack (Fig. or few 4F) have Nitzschia dubiiformis Nitzschia and e row of rounded poroids. A central larger larger Acentral ofroundedporoids. e row N. pellucida ities of fibulae and striae are 12-15 and 35-40 12-15 are ities offibulaeandstriae μ m) are solitary and possess two m) are solitary andpossess μ m) have two chloroplasts andcolonies m) havetwochloroplasts and Antarctic waters butalsoin waters Antarctic and from GenBank (sequence identity identity (sequence from GenBank Nitzschia (99.6 %sequence identity) and and Cylindrotheca N. pellucida N.pellucida

and

Accepted identity. by differ where 1.2%sequence (Fig. theirsequences phylogeny 2A) 1)andFig. thetwospec % sequenceidentity, 2016). compone possibly oneoftheendemic representing ofmorelightlySome striaeare composed simply 5-6poroidsin1 1 row poroids, ofrounded densities offibulaeandinterstr The (Fig. 4I). nodule bya central interrupted therapheishere and interspace have alarger are notalw Thefibulae pointed. ends are broadly (apical 26-60 axis: Article overlap cell colonies andeach as anewspecies, described regions (Table2). Pseudo-nitzschia granii isabsent. central interspace in10 fibulae (16-18 poroids entirely The (Fig.formed 4G). Pseudo-nitzschia arctica Pseudo-nitzschia arctica Four Pseudo-nitzschia arctica Pseudo-nitzschia Pseudo-nitzschia This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. μ m; transapical axis: 1.6-2.5 m; transapicalaxis: has been reported in northern cold waters, including Arctic and subarctic andsubarctic Arctic including cold waters, hasbeen in northern reported strains isolatedduringMALINA the seems to have a distribution c seems tohaveadistribution and &Percopo Sarno. Pseudo-nitzschia arctica Pseudo-nitzschia arctica s the next sibling cell forca1 sibling cell s thenext iae are 17-24 and 34-39 in10 and 34-39 iae are 17-24 P. granii share highly similar 18S rRNA gene sequences (99.6 sequences gene sharesimilar 18SrRNA highly μ m. Each poroid most often contains 1-6 sectors. 1-6 m. Eachporoidmostoftencontains ies can be better separated using 28SrRNA using ies canbebetterseparated μ ays regularly spaced. The two central fibulae two centralfibulae The spaced. ays regularly silicified areas without any perforations. silicified areaswithout any perforations. m) are lanceolate in valve view. The valve view.Thevalve invalve m) are lanceolate nts of the Arctic diatom flora (Percopo et al. (Percopo nts oftheArctic diatomflora (Percopo et al. 2016). Cellsoccur in et al.2016). (Percopo onfined to the northern polar area, onfined tothenorthernpolararea, ⁄ μ 8 of its length (Fig. 4H). Cells 8 ofitslength (Fig. 4H). μ m) are irregularly andthe spaced cruise have beenrecently cruisehave m, respectively. contain Thestriae from 2). (Table Arctic waters (Fig. 4,MandN). areolae thesurrounding intoaholelarger than rimoportula opens externally apices 4L). The (Fig. tw A singlerimoportulaislocated Hasle etal.1994). L-N)slightlyof 5-7slits(Fig. 4, fromth different apical fieldsare The composed (Fig. somepartsofthevalve 4K). inthe the apicesalternate and (22-23 in10 striae No(Fig. colonieswere Theuniseriate 4J). observed. %) and affiliated to belonging to AcceptedFragilaria to also related strainsare these from gene sequences from Article Cells (apical about55 axis: Synedropsis hyperborea Fragilariaceae. Fragilariaceae was not well resolved was notwell taxonomy Fragilariaceae Synedropsis hyperborea

S. hyperborea Synedropsis hyperboreoides Grammonema striatula species (Fig. 2A). 2A). species (Fig. This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. S. hyperborea S. hyperborea S. hyperborea S. hyperborea

strain RCC2043 shareshigh very fromGenbank similarities withasequence strain (99.9 %) (Grunow)&Hasle, Syvertsen. Medlin is typical of the Northern cold region and it is commonly reported in cold region reported anditiscommonly typical Northern is ofthe share identical 28S rRNA gene sequences and group with a sequence groupwith asequence and 28SrRNAgenesequences share identical μ (99.5%,Fig.MALINA strainsRCC2043andRCC2520 1). m; transapical axis: m; transapicalaxis: 2.7-3.5 from GenBank (98.5 % sequence identity). The 28SrRNA The identity). (98.5 %sequence from GenBank

as well as as well Synedra minuscula Synedra at reported in the original description (4-6 slits, description(4-6 at reportedintheoriginal based on 18S rRNA gene since the sequence thesequence on18SrRNAgene since based Thalassionema frauenfeldii o or three striae from one of thetwovalve fromone o orthreestriae μ m) are lanceolate invalveview m) are lanceolate

(99.9 %), (99.9 %), μ m) are parallel towards m) Fragilaria and

3 sp. (99.9 of thetwoMALINAgene stra from 1). The28SrRNA longicornis of ofsequences with that branches variable (12- Thelength rimoportula(Fig.of thehornsis 5C). the almost circular andlack are Valves are present. chloroplasts plate-like two and B).One or 5,A horn-like projections(Fig. waters (Table 2).The 18SrRNAge 2).The waters (Table strains (Fig.longitudinalE). 3or4inbothexamined 5,Dand stripscanbe The numberfrom ranges of length 2.9to4.4. cell diameter horn and theratio and between in acentral of several of strands Anumber fultopor 5F). (Fig. (Figs. discrimination ofthedifferent 1and2B). foundhere species Thalassiosira, Porosira, mantle. A single rimoportula (or labiate process) is locatedwithintheinnerring ofmarginal is rimoportula (ormantle. Asingle labiateprocess) and the the valve face of betweenthemargin rings placed toform3-4 arranged fultoportulae are

AcceptedA. septentrionalis Article Cells (apical 3.5- axis: Attheyaceae .

Cylindrical Thalassiosira gravida

This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. cluster and several fultoportulae are scattered on the valve face. Themarginal face. onthevalve are scattered fultoportulae cluster and several (99.8 %)

cells (diameter: 28.5-30.5 (diameter: cells .

Attheya septentrionalis Attheya septentrionalis

and from GenBank and is related to sequences from three from three tosequences GenBank andisrelated from

We isolated 12Thalassiosiraceae and A. longicornis 6.4 Cleve. Cleve. is distributed in the northern coldregion inArctic isdistributedinthenorthern anditiscommon μ Shionodiscus m; pervalvar axis: m; pervalvar axis: ne from strains theMALINA ne A. septentrionalis A. septentrionalis ( ≈ μ (Østrup) Crawford. 98 % sequence identity, Fig. 2A). identity, 98%sequence . Both 18S and28SrRNAgene. Both allowed the 18S m) held in colonies by a single thick threadcomposed thick coloniesbya single m) heldin 7-11.7) are solitary and bear four slightly 7-11.7) areand bear wavy solitary tulae (or strutted processes, 11-15) are grouped are 11-15) tulae (orstruttedprocesses, ins is also highly related to that of sequences tothatofsequences ins isalsohighly related (99.9 % sequence identity)and(99.9 %sequence

strains (Table 1) affiliated to the genera tothegenera affiliated (Table1) strains

RCC1986 andRCC2042 Biddulphia

Attheya Attheya spp. (Fig. 35 μ m) highly related to 2 sequences from to2sequences highly related from bothourstrainsof from both previously observed in Arctic and Antarctic waters (Table 2). (Table2). andAntarcticpreviously waters inArctic observed siliceousareas. radial perforated ribsseparate where in thecentralpart, valve (16-20in10 onthemarginsthe areolae ofthe are well-formed butingeneral ofsilicification, degree avariable valveshave Different fultoportulae (Fig. 5G). Accepted rim andone imperforated ba several open and a copula a valvocopula, isformed by girdle The thevalve(Fig. 5I). throughout Short andminutespineshairsemerge margin middle.Abroad ispresent. hyaline It tooneofthemorinthe either closer them. be can The rimoportulaispositionedslightly insideth tubes(Fig. Allthefultoportulaeexternal 5,H-K). fultoportulae Themarginal(Fig. havelong 5H). thevalvefultoportula are face on present fultoportulae (5in10 marginal ring of One andmantle. valve face (30in10 areolae The centralthread. by4 cells)connected one Article Thalassiosira The 18S rRNA gene sequence from from geneThe 18SrRNA sequence Thalassiosira gravida Cells (diameter: 6.5-13.5 Cells (diameter:6.5-13.5 T. gravida Thalassiosira eccentrica Thalassiosira eccentrica This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. and cf. T. rotula hispida T. gravida is regarded as a bipolar, cold to temperate water species andithasbeen is waterspecies totemperate regarded cold asabipolar, advalvar row ofareolae advalvar Syvertsen. Syvertsen. μ (sequence identity > 99.5 %) and is highly related with a sequence withasequence related 99.5 %)and ishighly (sequence > identity m) possess several chloroplasts, and form colonies of few cells (3- offew andformcolonies m) possessseveral chloroplasts, (99.3 % sequence identity, Fig. 1). The 28S rRNA gene sequences rRNA genesequences The 28S Fig. 1). identity, (99.3 %sequence T. rotula group with two other sequences from from group othersequences withtwo T. gravida nds. The valvocopula has a broad abvalvar abvalvar abroad valvocopulahas nds. The (99.2 % sequence identity, Fig. 2B). 2B). Fig. identity, %sequence (99.2 e ring of marginal fultoportulae, between twoof between ofmarginale ring fultoportulae,

have four satellite pores at their base (Fig. 5I).at their base(Fig. pores have foursatellite strain RCC1984 clusters with sequences from strain RCC1984clusterswithsequencesfrom (Fig. 5J). MALINA(Fig. of 5J). strain μ m) have a similar size on both size a similar m) have μ m) and one central onecentral m) and μ m) andpoorly developed T. gravida T. cf. and are and are hispida is T. nordenskioeldii from RCC2521clusterswithsequences ourstrain of sequence 24- (18 and number ofareolae morphologically of similartotheoriginal very description (Fig. 5, M and N). Each marginal fultoportula is accompanied with a small external labiate- external withasmall marginal fultoportulaisaccompanied Each (Fig. 5,MandN). slightly tooneofthem twomarginal fultoportulae,large rimoportulaisplacedbetween closer occasionall Five havebeen fultoportulae and N). in 10 in 10 (30-35 The areolae (Fig. 5L). inthecentre depressed slightly valve face and witha cell diameter the with apervalvar generally axis In shorter than rectangular conditions. girdle cellsare view, Acceptedfrom RCC2521 and sequence identity) Article from rRNA genesequences edge. awidenon-pierced with andavalvocopula a broad onthevalve hyaline of margin the presence specific for isnot which however 1986). Very onthevalvesurface, similaristhedense covering ofspinules Cells (diameter:4.5-13 Thalassiosira minima Thalassiosira hispida μ μ m) with short external tubes and one or two central fultoportulae are present (Fig. 5,M present (Fig. are centralfultoportulae two m) withshortexternal tubesandone or fultoportulae(4-6 marginal ringOn thevalve,ain shape(Fig. of 5M). m) are hexagonal This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. T. hispida

(Fig. groups 2B) with (96.5 %) but the clade is poorly (< 50 % ML and NJ) supported. supported. NJ) ispoorly(96.5 %) and 50%ML buttheclade (< , but can be developed to a lesser extent inother extent toalesser , butcan bedeveloped Thalassiosira angulata has only been reported in northern cold water regions (Table 2). 18S (Table2). cold regions has water innorthern only reported been Gaarder. μ m) have two chloroplasts and do not form colonies under our culture under our culture anddonotformcolonies m) havetwochloroplasts T. hispida 26 in10 T. allenii are not available on the GenBank and the18SrRNAgeneare not availableontheGenBank and μ m on valve face and mantle, respectively, in Syvertsen respectively, inSyvertsen andmantle, m onvalveface (98.6 %, Fig. 1). The 28S rRNA gene sequence (98.6 28SrRNAgenesequence %,Fig. 1).The (97.5 %) (97.5 yA observed valve (Fig. inonesingle 5O). ,

Thalassiosira aestivalis T. hispida Thalassiosira allenii but possesses ahigher Thalassiosira (97.1 %) (98.5 % species, and

and RCC2000 shares identical 28S rRNA gene sequence with another RCC2000 shares 28SrRNAgene identical sequence and common in Arctic waters (Table 2). (Table2). common inArcticwaters fultoportulae B (Fig. C). and 6, positioned withintwomarginal fultoportulaandonerimoportula terminalcentral collar, one in10 oblique mantle,amarginal fultoportulae ring of (3-4 6B). and18-20in10 face 17-18onvalve are Areolae (Fig. 6A). thread a central strains. phylogeny, the18SrRNAgene(Fig. clade Consistentwith strain CCMP990forming 2B). awell-supported group (99.1%)and withthe theAntarctic strainRCC2707 with rRNA genesequences highly similar28S share (Table 1) Ocean North Pacific andthe from boththeBeaufort Sea the Accepted Article for Ocean. thefirsttimeinArctic Thespecies aworl has shaped 5P). protrusion(Fig. The 18S rRNA gene sequence from stra from geneThe 18SrRNA sequence nordenskioeldiiThalassiosira Cells (diameter: 12-15 Thalassiosira nordenskioeldii The 18S rRNA gene sequence from our from geneThe 18SrRNA sequence T. minima T. nordenskioeldii

Valves are characterized by a pronounced concavity in the centre, a high (4-6 areolae) and areolae) (4-6 ahigh centre, inthe concavity by apronounced characterized are Valves Thalassiosiracurviseriata This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. sequence from thestrainfrom CCMP990 of Our strains sequence (99.7%,Fig. 1). forming a well-supported clade (Fig. 1). (Fig.clade 1). forming well-supported a μ m) possess several chloroplasts and form long colonies connectedby andformlongcolonies chloroplasts m) possessseveral is a Cleve.

species typical of northerncoldspecies totemperate regions, typical is the species most closely related to all the mostclosely to is thespecies related in RCC2000 groups with sequences from in RCC2000groups withsequences T. minima T. minima dwide distribution (Table 2) and it is reported dwide distribution(Table2)anditisreported strain RCC2265ishighly similartothatof μ m) with long external tubesbearing a m) withlong external Thalassiosira nordenskioeldii T. nordenskioeldii T. nordenskioeldii μ m onmantle(Fig. T. minima T. minima T. aestivalis strain from T. minima the GenBank and highly similar 28S sequence (99.8 %)similar 28Ssequence with (99.8 the GenBankandhighly strains form a clade with a sequencefrom with strains formaclade (3-4 in10 Dand Fig.colonies (2-3cells; E).Numerous 6, oestrupii from sequences 18S and28SrRNAgene CCMP1099andRCC2709(Fig. Antarcticstrains (99.6 %), tothatofthetwo 2B). fromtheMALINA28S rRNAgene stra sequence 6F). is annulus ispresentanda rimoportulaprocess large Accepted of StrainRCC1991extensions. isthefirstrepresentative fultoportulae (4-7 marginal I). Hand rimoportula(Figs. a subcentral The 6, valve centresingle and fultoportulainthe areolati andthemantleisrounded.The convex isgenerallyaxis longer pervalvar (Fig. 6G). The Article Cells (diameter: 30-40 Porosira glacialis Cells (diameter:23-41 Shionodiscus bioculatus Porosira glacialis RCC2039 and μ This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. m). The striae (24-27 areolae in10 m). The (24-27areolae striae

18S rRNA is identical with that from the Antarctic strain CCMP1099(Fig.18S rRNAisidenticalwiththatfromtheAntarctic 1) strain Shionodiscus ritscheri (Grunow) Jørgensen. (Grunow) Jørgensen. is reported in Arctic and An is reportedinArctic μ μ m) are cylindrical, possess several chloroplasts and canform short and severalchloroplasts cylindrical,m) are possess m) are solitary and possess a large number of discoid chloroplasts of alarge discoid number m) areand possess solitary (Grunow) μ m apart) have internal tube-like projections and no external no external internaltube-likeprojections and m apart) have (Figs. 1 and 2B). 2B). (Figs. 1and

Alverson, Kang & Alverson, Kang Theriot. this strain group with sequences of of this straingroupwith sequences T. aestivalis T. aestivalis on is fasciculate (20-23 areolae in 10 in10 areolae (20-23 fasciculate on is μ fultoportulae are scattered over the valve surface thevalvesurface over scattered are fultoportulae than the diameter. The valve faceisslightly The valve the diameter. than m) are wavy and radially arranged. Acentral arranged. m) are andradially wavy tarctic waters (Table 2). 2). waters (Table tarctic in RCC2039ishighlyrelated, butnot identical situatedinsidethemargin (Fig. ofthevalve (Fig. 2B). S. bioculatus T. nordenskioeldii sequenced to date, both sequenced todate, Shionodiscus RCC2021.These μ m) witha . The The belgica familynamely fromthe representatives , thatofother with cladeNJ) whichbranches 100 % (96%ML, both formawell-supported from totwosequences highly related AcceptedMinutocellulus including 2) ArcticOcean(Table pilus base(Fig. 7A). Patch in processvalves. seem tobemorepronounced Articlecan be which smooth orornamentedbycostae onthe can bepresent of poroids variable number a valveandmargin ofthe ofporoidsisalways along present row and 7A).Amarginal the Thepro (Fig. 6J). conspicuous branches and bear cross each other L). (Fig. 6,Kand Thepili andK).Eachvalvehas cells (Fig. twoocelluli 6,J apilivalve,whichar and valve valves, aprocess 1.7-2.2 axis: 18S phylogeny could not discriminate 18S phylogeny couldnot cornucervis Arcocellulus Cells are 3.0 (apical solitary, very axis: small cornucervis. Arcocellulus Cymatosiraceae. , and This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. Brockmanniella brockmanni Brockmanniella μ m) and slightly curved in broad girdle view. Each cell possesses two different twodifferent cell possesses view.Eachgirdle inbroad m) andslightly curved (Fig. 1). The 18S rRNA gene sequence from from geneThe 18SrRNA (Fig. sequence 1). has been reported in temperate and cold waters ofbothhemispheres, and coldwaters has been intemperate reported Hasle, von Stosch &Syvertsen. Hasle, vonStosch&Syvertsen. . Minutocellus polymorphus Minutocellus Arcocellulus Arcocellulus (Fig. 1). indistinct or more convoluted (Fig. 6K). Costae ormoreconvoluted(Fig. 6K). indistinct – e convex and concave, respectively, in larger inlarger respectively, andconcave, convex e cess valve possesses a central process (Figs. 6K process(Figs. a central valvepossesses cess 3.5 valve face. The basal siliceous layer may basalsiliceouslayer be The valve face. μ es of short spinules can be present near the bepresentes ofshortspinulescan near m; pervalvar axis: 1.4-1.7 1.4-1.7 m; pervalvar axis: spp. fromtheclosely relatedgenus elegans Papiliocellulus A. cornucervis A. cornucervis (99.5 % sequence identity) and identity) (99.5%sequence RCC2270 , μ m; transapical

is indeed respectively, and 4 other being closely genotypes related affiliated to genotypes, 2ofthemcorresponding tothespecies into6 groupedITS C)we strains phylogeny and 3,Band (Fig. these (Fig. and 3A) 2B, Fig. 2B). identity, (96.9 %sequence our genefrom clade 1).The28SrRNA (Fig. of 2). waters(Table cold typical northern ofthe valve (Fig. 7C). the of onthecentre Arimoportulaispresent apertures (Fig. 7B). view withsquare tohexagonal chloroplasts. Cellsformcolonieswhichcan several (Fig. 2B). and 95.9%sequ unidentified Cymatosiraceae (95

AcceptedEucampia zodiacus Article Chaetoceros decipiens The 28S rRNA gene The 28SrRNA of . Chaetocerotaceae. from geneThe 18SrRNA sequence Eucampia groenlandica Cells (apical 7-24 axis: Eucampia groenlandica . This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. (99.2 %) (99.2 We isolated 45 strains of the genus isolated45 strainsofthe We μ A. cornucervis A. Cleve was first reported from Baffin from Baywas firstreported Cleve. m) are rectangular in girdle view, slightly silicified andpossess girdle view,slightlym) are in silicified rectangular

and . Eucampia antarctica Eucampia E. groenlandica E. groenlandica strain RCC2270 is closely related to thatoftwo RCC2270 strain to isclosely related strains is related to a sequence from isrelated toasequence strains ence identity) isolated ence identity) Chaetoceros decipiens be straight or slightly curved in broad girdle inbroad orslightlybe straight curved strain RCC1996groups withsequences (99.0 %) forming (99.0%) awell-supported Chaetoceros in Davis Strait and isconsidered in DavisStrait C. neogracilis and using the28SrRNA andusing from temperatewaters from and and C. gelidus E. zodiacus .

side. oneach largesingle pores longitudinal rowof section,withspinesontheedgesa mostly and arefour-sided, in cross polygonal, The setae with parallel transverse interspaced costae by are ornamented (Fig. bands 7H). projection Girdle small centralprocesswithashortexternal possessavery (Fig. 7F).Terminalvalves and mantle valveface the ridge between ispresent irregular ribs radiate andare perforated with annulusfrom which central G).F Valves a mantle, isalmostflatingirdleview(Fig. and have 7, withahigh Thevalve, 7,Dand E). (Fig. UorVshaped setaeare Terminal longer distance. from wi thevalvemargin emerge intercalary setae ellipt are apertures andthe Chains arestraight possessesseveral 7,Dand cell chloroplasts. (Fig. observed E).Each have been Accepted withasmall In tooval,ingirdle7L). itisslightly iscircular concave valveview,the view forming apertures (Fig. hexagonal narrow after apart the valve shortbasal margin andmerge emerge inside setae The group colony(Fig. Several together forming 7K). chains aspherical 7J). lorenzianus from GenBanksimilarly, sequences to gene the28SrRNA isclosely related from GenBank sequence Article Cells (apical 11-22 axis: The 18S rRNA gene sequence from ourstrainof from geneThe 18SrRNA sequence Chaetoceros decipiens Cells (apical 4-12 axis. Chaetoceros gelidus This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. (99.2 %) , and groups with and Chamnansinp, Li,Lundholm Chamnansinp, & Moestrup. Chaetoceros is a cosmopolitan species, common in arctic waters (Table 2). (Table 2). common inarcticwaters acosmopolitanspecies, is μ μ m) with a single lobed chloroplast are joined in curved chains (Fig. are chains(Fig. joinedincurved chloroplast m) withasingle lobed m) were generallym) wereconditions butafewcolonies solitary inculture cf. Chaetoceros affinis Chaetoceros lorenzianus lorenzianus small poroids.Themantleishigha marginal and ical. All setae lie in the apical plane.The All setaelieintheapical ical. hyaline areas with scattered small poroids (Fig. 7I). 7I). poroids(Fig. areas withscattered small hyaline thout a basal part and may fuse for a shorter or a shorter or for fuse thout abasal partandmay C. decipiens C. (97.1 % sequence similarity, Fig. 1) similarity, (97.1 %sequence and Chaetoceros diadema (RCC1997) Chaetoceros

groups witha

(Fig. 2B). (Fig. 2B).

and, gelidus morphology Variability etal.2013)isabsenthere. inspore of description (Chamnansinp reportedintheoriginal crest from andsmooth.(Fig. eachresting 8C).The are convex spore between (Fig.Both moredistant distal partitpossessesspineswhichare other each valves 8B). spines onitsbasalexhibit onits part, whereas not length.Incontrast setadoes straight thelong its throughout spinesoccurring arranged densely spirally setaehave short long seta.The straight setaeandone shortcurved cellshavethree the Generally (Figs. and8A). 7L central inflexion Accepted ellipticallyfrom varying byapertures separated andthey are joined toformstraightchains ce to thedifferent variability beassociated might butthis D-F), setae(Fig. 8, curved andothers axis, havemore perpendicular topervalvar setaediverging at some strainshavestraight oface strains, withtheexception among thedifferent observed andultrastructural been has difference significant morphological 4-12 relatively axis: small(apical have beenoccasi but shortcolonies(3-6cells) solitary generally 8,D-F) (Fig. using1).Cellsare examined EM(Table further strains havebeen formostofthem(htt photographs available are (Fig. 2B). Article tothat ofthetype strainof identical are identity,and 28SrRNAsequences Fig. 1) The 18S sequence of The 18Ssequence (Table 2). Ocean waters, cold from including Arctic reported The species been northern has Twenty-eight of ofthe36strains Chaetoceros neogracilis was already reported (Degerlund et al. 2012, thereinas etal.2012, (Degerlund was reported already This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. C. gelidus (Schütt) VanLandingham. (Schütt) VanLandingham. rtain variability in theorientationofsetae. cells, Assingle in rtain variability μ clusters with a sequence of clusters withasequence m) and possess a single lobed chloroplast (Fig. 8, D-G). No (Fig. D-G). chloroplast m) andpossessasingle 8, lobed C.neogracilis an angle of 45°, whereas others have setae havesetae others whereas of45°, an angle onally observed (Fig. 8G) in 9 strains. Cellsareonally 8G) in (Fig. observed p://www.roscoff-culture-collection.org). Seven p://www.roscoff-culture-collection.org). ll sizes of the strains. In colonies,cells ofthestrains. sizes are the ll isolated here C. socialis C. socialis C. socialis

have been observed by LM by and observed been have , northern strains). , northernstrains). (97.2 % sequence (97.2 %sequence C. gelidus C. indiscriminately to many small,unicellularindiscriminately tomany the testedstrains. M andN). observed(Fig. Spores inany 8, were of costae not by shorttransverse interconnected Accepted Antarctic strain from the with thesequence clade a well-supported ArM0005 andform e strainsArM0004 with thetwoArctic sequence for strainsRCC2016andRCC2318.ThesetwoMALINA gene has been Thefull18SrRNA sequenced (data notshown). gene V4 region ofthe18SrRNA well asAntarcticregions. co component ofmicrobial as asignificant reported name of thespeciesis correct word andthe as amasculine thegenus iscurrently recognised than masculine.However gracile epith Thespecific aHargraves discussion). 1988for Articlel (about 2spinesper spines with arrowhead-shaped spiral costae ornamented circular setaeare L).and The directions (Fig. 8,H margin indifferentand divergerunning chain the valve atthe cross apices, immediately andnotcells indi the chains arerealcolonies isabsent inthe process I The central 8, and J). theannu in thecentreof slit-like process islocated Inthe terminalvalves,a annulus. acentral from costae originating withirregularare ornamented Valves or Vshaped. U are Terminal setae to narrow slits(notshown). shaped G and (Fig. H) 8, The name All the or C. neogracile C. neogracilis This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. C. neogracilis C. neogracilis , because the genus, because the strains isolated during the MALINA cruise share 100% identity inthe strains isolatedduringMALINA share100% cruise the (basionym: . In more recent years, the species has been consistently been consistently years,In has thespecies . recent more C. gracile AnM0002 (98.9 % sequence identity, Fig. 1). The The Fig. 1). identity, (98.9% sequence AnM0002 Chaetoceros Chaetoceros Chaetoceros vision (Fig. 8K). Intercalary setae originate from originate Intercalary setae vision (Fig. 8K). tercalary valves of the colonies, confirming that confirming that colonies, valvesofthe tercalary in cross section. They are composed by long composedbylong incrosssection.They are lus and it bears an external flattened tube (Fig. flattenedlus anditbearsan tube external Schütt) hasSchütt) almost been attributed mmunities in Arctic and Baltic (Table 2) as Baltic 2) (Table inArcticand mmunities et can be found in the literature spelled as spelled et canbefoundintheliterature taxa collected worldwide (see Rines & (seeRines collectedworldwide taxa was considered to be neutral, rather rather beneutral, consideredwas to strains share identical 18SrRNAgene strainsshare identical μ m) and C. Chaetoceros tenuissimus Antarctic with branch strains(CCMP163,CCMP189andCCMP190).Allthesesequences (Chamnansinp etal.2013) strainBaltic CPH9attributedfrom to the sequence as wellwithaGenBank from fromseparated fallsII withinCladethe Antarctic and 3A).ThestrainCPH9 inbothML (Fig. IV andNJ support (>Clade 50%) emerge withmoderate II fromCladeare strains clades. Specifically One group consists of neogracilis the (Fig.phylogeny 3A)separates gene 28SrRNA 1). The IV and 6toClade (Table I,BITSbelong(Figs.II, andC,S1),20strainsIII 8toClade toClade 3, phylogeny 2toClade and (Fig. 3A) oneitheror both28SrRNA based used.Overall, markers based onallthethree ITS-2,of 5.8S + respectively. strains TheMALINA Chaetoceros theArctic example For branches. and theAntarctic strains Arctic between the ITSup to0.5%.Both as well28S markers shared similaralthough highly notidentical 28S of strains fromtheMALINA 28S rRNAgene sequences AcceptedIII,CladeIV. and 6strains toClade Strains from Article Genetic of diversity C. neogracilis This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. strains in two groups, both with high (> 75 % in both ML and NJ) bootstrap support. NJ) strains intwogroups, 75%inbothMLand (> bothwithhigh sp. CCMP189 95 %, 86 %, and 85 % sequence similarity for the 28S, ITS-1, and ITS-1, 28S, sp. CCMP189 forthe sequencesimilarity 95%,86and85% C. neogracilis , with 15 of them forming Clade I,II,, with15ofthemforming Clade 4strains Clade to belonging 2strains to C. neogracilis Chaetoceros neogracilis Chaetoceros forming a well-supported clade (Fig. 3A). clade 3A). (Fig. forminga well-supported

and have a sister clade whichin a sisterclade and have . Both ITS-1 and 5.8S + ITS-2 trees includes 27 Arctic sequences ITS-227 Arcticsequences ITS-1 includes + . Both trees and5.8S C. neogracilis C. neogracilis Clade I, whereas the second group theotherI, 3 Clade includes thesecond whereas strains CCMP163,CCMP189and strains CCMP190 are fully

(Fig. 3), since the two groups form two separate two separate form groups3), sincethetwo (Fig. at the base of the group from which Clade IIIgroupand whichClade from at thebaseof rRNA gene indicate significant differences significant geneindicate rRNA strains. rRNA gene sequence. Sequences can diverge by divergeby can Sequences genesequence. rRNA RCC2014 shares withtheAntarcticstrain each clade cluster between them withmoderate clusterbetween clade each C. neogracilis The MALINA strains of The MALINA strains C. neogracilis cludes the sequences from three from cludes thesequences form four different clades clades different form four cluster together (Fig. 2B) clustertogether(Fig. C. fallax C. neogracilis C. strains of strainsII fromClade thedifferent 3C). (Fig. between I occur and somedifferences IV mo are I andClade Clade supported, whereas IIIII and Cladeare highly phylogenyIn Clade bootstrap support(Fig. 3B). 5.8S+ITS-2 ITS-1II, support in Clade phylogeny andClade approaches during the last decade revealed a cons revealed a decade thelast during approaches theirmorphology(G on solely described based Accepted diatomsf ofcultured characterization the cruise allowed MALINA strainsisolatedduring the onphytoplankton approaches DISCUSSION 9). (Fig. clades theotherthree (GU) towards c III versus and clade (AU) IIa helix between (CG for 2nucleotides.Twohemi- inHemi-CBC for6nucleotides,andCBC the helices. inpairedpositionsconsists Thisvariability pairedpositionsof strains at14positions,9ofthemlocatedin occur ITS-2 fromour sequences ArticleIII IIa located(Fig. inthe (IIb) helix Differences as anadditional between andhelix 9). helix 2009)aswell (Coleman ofalleukaryotes IV)IIa,III typical fourhelices(I, and strains exhibits (2009). toColeman according secondary structure inpositionspairedthehelicesof CompensatoryBase(CBC) ChangesHemi-CBC and Secondary structure of ITS-2. Secondary structureofITS-2. Combining microscopy and geneticdata. Combining microscopyand ↔ UG, GC UG,GC C. neogracilis This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. ↔ GU, GU GU, to further investigate their genetic differences. We determined genetic We differences. their to furtherinvestigate CBC occur in helix I CBC (GC occur inhelix ↔ AU), and 1 in helix IV (GU and 1inhelix AU), We predicted the secondary structure of ITS-2 rRNAforour ITS-2 of Wepredicted structure thesecondary rom the Beaufort Sea. To date about 10 about Sea. Todate rom theBeaufort

The combination of morphological and molecular ofmorphological The combination lade IV (GC), with clade III showing a Hemi-CBC Hemi-CBC III a showing IV (GC),withclade lade uiry 2012) and theapplication ofmolecular uiry2012) III, and Clade IV group withtogether high derately supported; Clade III groups withClade supported;Clade derately iderable genetic diversity within key genetic diversity planktonic iderable The secondary structure of ITS-2 fromour ITS-2 of structure The secondary ↔ AC,andCG ↔ GC). Moreover 1CBC on occurs GC). Moreover ↔ UG), 3 in helix III UG), 3inhelix 4 species havebeen

et al. 2006, Alverson et al. 2007, Hoppenrath et al. 2007) and here they provided a good a they provided 2007)andhere 2007,Hoppenrathetal. al. et al.2006,Alverson identificationof forgenes thetaxonomic used arecommonly et al.2011)andthe genus, depending on the phylogenetic distan phylogenetic genus, onthe depending genus tothe withinagiven wasaccording investigated,used here different butitalsovaried Sea. clarified of theidentity 36 genetic rRNAdiversity of investigatedin GenBank. Finally, we the areasthatareavailable geographic from different fr strains wereobtained from different theArctic arctica groenlandica, S.bioculatus, first timefor6diatomspecies( forthe sequenced here morphological information and ultrastructural exceeds by one order of magnitude of thosede order by one exceeds diatomspecies It thatthenumberof extant 2007, Kooistra beensuggested etal.2008). has al. 2013),and Lim et etal.2013,Orive Lundholm(LundholmLundholm etal.2012, al. 2003, etal.2006,Amato& 2008, Montresor et Leptocylindrus danicus Accepted Article as morphospecies such The 18S rRNA gene can successfully discriminate species within the genus speciessuccessfully discriminate withinthe genecan The 18SrRNA Genetic delimitation. markersandspecies Our both18Sand28SrRNA work provides

has been also sequenced for the first time. Moreover, most of the gene sequences sequences gene ofthe Moreover, most thefirsttime. for also sequenced has been This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. C. closterium C. closterium Asterionellopsis C. neogracilis C. Cleve (Nanjappa etal.2013) and Chaetoceros T. species complex (Haitao 18Sand28SrRNA etal.2007).Both species complex cf. , a taxon that , ataxon hispida costatum Skeletonema

glacialis strains sharing the same 18S genesharing same sequence 18S strains the scribed to date (Mann & 2013). Vanormelingen (Mann todate scribed ce existing between congeneric species. species. congeneric between existing ce ). The 18Sgene). The of The taxonomic resolutionofthe The taxonomic geneticmarkers for 17 morphotypes. Both genes have been genes havebeen Both17 morphotypes. for gene sequences vali om sequences from conspecific strains collected strains conspecific sequences from om (Castracane) Round (Kaczmarska et al.2014), Round(Kaczmarska (Castracane)

dominated genetic libraries from the Beaufort fromtheBeaufort dominated genetic libraries , Pseudo-nitzschia pseudodelicatissima , Pseudo-nitzschiapseudodelicatissima A. cornucervis, C. decipiens, E. C.decipiens, A. cornucervis, Thalassiosira (Sarno et al. 2005,Sarnoet (Sarno al. et C. gelidus dated with detailed dated with , species (Kaczmarska N. pellucida Nitzschia , and (Rimet , and

P. and bebetter 1)butcan (Fig. rRNA genesequences 2 are involved in ribosome assembly (Tschochner & Hurt 2003) and changes in paired positions changes inpaired & 2003)and Hurt (Tschochner assembly2 are involvedinribosome ITS-ITS-1and both of secondary structures The (Coleman 2009). tointerbreed clades unable are IIIa asbetweenClade II, aswell Iand Clade IV, at of and presence II similarlyvs. clade the IIaSpecifically aCBC 9)suggests (Fig. inhelix of four clades thisgroupingconfirm andwo secondary structure neogracilis ITS28S and gr consistently rRNAphylogenies genetically(Fig.are differ 1),butthey sequences andEU090014; Choi (EU090013 Greenland Sea al. 2016). et al.2013,Percopo et Lundholm ITS2Lim etal.2013,Orive the 2003,Amatoetal.2007, etal.2012, (Lundholm etal. ITSwith the rRNApossiblyachieved supplemented geneshares with identical18SrRNA Accepted Articlenitzschia (Whittaker al.2012). et ITSsequencing after onlyseparated also on28SrRNAge distances phylogenetic foralltheTha resolution taxonomic Similarly,strains of theMALINA The 28S rRNA gene is a relatively good molecular marker to discriminate most of genegood markerrelativelyThe 28SrRNA todiscriminate molecular isa rbc L phylogenies (Percopo et al. 2016). L et (Percopo phylogenies species althoughabetterresolutionofphyl into four phylogenetically discrete clad discrete intofourphylogenetically This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. C. neogracilis C. neogracilis which may correspond to closely related but distinct cryptic tocloselywhich may butdistinct related species. correspond Pseudo-nitzschia arctica lassiosiraceae representatives except except representatives lassiosiraceae C. neogracilis T. rotula

ne phylogeny (Fig. 2B) and can be correctly becorrectly can and 2B) (Fig. phylogeny ne nd all the other clades, suggests that the different thatthedifferent suggests theotherclades, nd all (Fig. 1). These two species show low twospecies (Fig. 1).These ouped sequences from the Arctic strainsof fromthe sequences ouped least a Hemi-CBC in the Helix III Clade intheHelix between least aHemi-CBC discriminated based on 28S rRNA (Fig. 2A), ITS rRNA(Fig. 2A), based on28S discriminated reproductive isolation between clade Iclade and clade isolation between reproductive ent at both 28S and ITS atboth28Sand ent (Figs.and 3). 2B, levels et al. 2008) share identical 18SrRNA et al.2008)shareidentical uld indicate reproductive isolationbetweenthe uld indicate reproductive es (Fig. 3). The differences in the ITS inthe differences es (Fig. 3).The and two Arctic strains isolatedfrom the strains andtwoArctic ogenetic relationships can be generally generally canbe relationships ogenetic by the analysis of the secondary structure of of secondary structure ofthe analysis by the and

P. granii share T. gravida

highly similar 18S , which Pseudo- C. different species. For example, For example, different species. (Amato etal.2007). Accepted with gene sequences Lovejoygenotype etal.2008, a unique & Potvin2011). (Pawlowski clades among of four et al.2015),failedtodiscriminate the Logares Logares etal.2014,Balzano Comeaual. 2012, etal. 2011, et studies (Stoecketal.2010, V9 regions ofthe18S V4 or ofthe sequencing etal.2012b). (Balzano cruise Sea during theMALINA throughoutBeaufort the al.2012a),which et ITSgenotype (Balzano contrast, theArctic catenella ArticleAlexandrium tamarense ITS genotypesfrom . Several thesameenvironment in co-occur genotypes can of genotypes thandifferentrather interbreed speciesunableto hypothesis thatthey beconsideredgives supporttothe should separate further ITS-2 inthe within the differing by CBCHemi-CBC or inability betweenstrains diatoms, (Coleman 2001).For hasbeen tointerbreed demonstrated likely gametecompatibility affects ce preventing Both 18S and 28S rRNA genesBoth aretooconserved 18Sand28SrRNA of ofdistinctgenetic clades occurrence The sympatric Notably, clone libraries based on18SrRNA clone libraries Notably, (Whedon & Kofoid) Balech Kofoid) (Whedon& This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. Micromonas Micromonas

A. longicornis A. longicornis (Lebour) Balech, (Lebour) A. septentrionalis (Lovejoy et al. 2007, Balzano et al. 2012b) consistedinasingle Balzano etal.2012b) (Lovejoy etal.2007, (Figs 1, and 2A). These two species can bedistinguishedcan These twospecies (Figs 1,and2A). , co-occurred in the Chukchi Sea (Gu et al. 2013). In etal.2013). (Gu intheChukchiSea co-occurred Alexandrium fundyense Alexandrium dominated both surface and DCM, waters DCM, and bothsurface dominated the Atama complex, which consisted of whichconsisted complex, Atama the a single species. Closely-related speciesor Closely-related a species. single rRNA, which are widely used inenvironmental used widely whichare rRNA, gene sequences, and high throughput amplicon and high sequences, gene lls differing by CBC or Hemi-CBC frommating lls differingHemi-CBC or by CBC and similar results were found previously found in and similarresultswere shared 18SrRNAand28S identical for some genera failing to discriminatethe failing somegenera to for P. pseudodelicatissima C. neogracilis C. neogracilis Balech

in theBeaufort Sea , and recovered them as andrecovered them and and Alexandrium species complex

from cornucervis 2012). ofseveralonly nucleara using acombination Acceptedgeneral studies onmicrobialeukaryotes.for can beused studiesfocusedon of alignment,for environmental comprom technologies the28S rRNAseemsthebest currentas 5,000bp(Mikheyev& Forreads sequencing Tin2014,Schlossetal.2016). aslong of thesequencing such asPacBiostudies. Single couldallow technologiessequencing molecule a resolutionandprovide taxonomic taxonomic from theentire rRNAoperon sequencing Ideally diatom species. of number for alarger available but28Ssequences are species congeneric thanthe18SrRNA rRNA geneislessconserved di genera differentmakes between thealignment dateinGenBank comp available to are sequences ITS 2015),very few & 2010,Guoet(Moniz al. Kaczmarska diatoms for as auniversalbarcode ArticleCymatosiraceae strains(Fig. 2B). and fromthisfamily isavailableonGenBank Cymatosiraceae noothersequence isnotclearsince gene withinthe ofthevariability the28SrRNA Theextent of (Luddingtonal. 2012). et The 18S rRNA gene is highly conserved also gene conserved The 18SrRNA ishighly Overall, ITS-2 provides a higher taxonomic resolutionthan28S,butalthoughOverall, higherITS-2 a taxonomic itwas provides proposed A. cornucervis M. polymorphus

This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. strain RCC2270 strain 18SrRNAwithtwoGenBank shares sequences almostidentical RCC2270 shares highly similar 28S rRNA gene with two unidentified gene RCC2270shares withtwounidentified similar28SrRNA highly (Fig. 1), and the 2 species share 100% identity in the V4region inthe 100%identity and the2speciesshare 1), (Fig.

nd plastidial encoded genes (Sorhannus & Fox Fox & (Sorhannus genes encoded nd plastidial nnotation frommostspeciesinenvironmental withinthefamily where Cymatosiraceae, the same specimen would allowthebest would samespecimen the fficult or even impossible. Similarly oreven fficult impossible. the28S allowing a better discriminationbetween allowing abetter ared to 18S and 28S and its highared to18Sand28S variability diatoms, whereas 18S rRNA gene sequencing diatoms,whereas genesequencing 18SrRNA ise between resolutive power and easiness and easiness powerresolutive ise between

A. 2015) with al. 2012b,Coupelet et (Balzano microphytoplankton cruise during theMALINA identified during late summer 2009 and during 2009 latesummer phytoplankton community. Clearly, by counts(http://malina.obs-vlfr. microscopy nordenskioeldii, T.gravida 2012b)andonly etal. (Balzano during theMALINA cruise sorted photosyntheticeukaryotes associated with environmental ribotypes al.2009). (Booth etal.2002,Sukhanova forming spring blooms 2002), eventually 199 &(Boothphytoplankton Horner assemblages typically they dominate where Arctic waters frequently in genera observed are from thesetwo first time in this study, might have been wrongly identified as the freshwater species first timeinthisstudy, asthefreshwater wrongly have identified might been Chaetoceros. as species, such Acceptedthat cellsof of reportedmorphotypes, theoccurrence butbarely of phytoplankton abundance thehigh countsconfirmed bygenetic assemblages, shown already the neogracilis present inlowabundance. Article Diatoms intheBeaufortSea. In spite of the high diversity reported in previous studies (Sukhanova et al. 2009),only few (Sukhanovaetal. studies In inprevious reported thehigh diversity spiteof The high number ofThe high number This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. , Chaetoceros

C. neogracilis reflected the dominance of these two species in the summer phytoplankton twospeciesinthesummerphytoplankton dominance ofthese reflected the We alsosuggestWe chainsof thatcell C. tenuissimus Chaetoceros and

might different have erroneously attributedtoseveral been solitary , Thalassiosira pacifica Thalassiosira T. gravida, or Diatoms represented an important fraction of the nano- and of thenano- animportantfraction Diatoms represented Chaetoceros simplex Thalassiosira strains (45), mostlyrepresented by strains (45), T. nordenskioeldii T. nordenskioeldii being the most represented genera. Different being species themostrepresented libraries (Balzano et al. 2012b). Notably, (Balzano etal.2012b). libraries cf. fr), accounting for a low proportion ofthe fr), accountingfor alowproportion hispida, C. neogracilis 7, Lovejoy et al. 2002, Ratkova &Lovejoy Wassmann et al.2002,Ratkova 7, species didnotbloominthe Beaufort Sea and few undetermined species were observed and few undetermined C. neogracilis Ostenfeld, or other undetermined orother Ostenfeld, C. gelidus and wereby among detected T-RFLP T. minima , which were described for the for, whichwere the described . Thisdiscrepancy indicates and other unidentified and other were possibly only C. gelidus C. gelidus and

C. T. 1998). from Danishwaters attributedto culture material this study because they might be more difficult to bring into culture compared to compared tobring culture into might bemoredifficult they studythis because ultrastructural similaritybe indeed could referand Danish to waters discussion on AcceptedOne ofthe seasonal salinity by shifts environments affected discerned. etal.2012b)cannotbe (Balzano MALINA cruise fromthe contribution totheenvironmental samples relative and thereforeribotypes their of clades different the Unfortunately, concentrations. might salinities, to lower higher beadapted ir ecological preferences indicativeof patterns are (http://malina.obs-vlfr.fr/data.html) compared innutrients and poorer saline(TableS1), waterswarmer, less surface MALINA were cruise gelidus IVisolated fromsurfa strains ofClade were Article al. 2012b). (Balzano et and inT-RFLP analyses C. neogracilis, Chaetoceros wighamii Other colonial Notably, some of thestrainsisolatedhereNotably, someof Interestingly mostofthe

and both C. neogracilis C. neogracilis This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. C. wighamii or because they are rare, as suggested by their absence in the 18S rRNA libraries libraries rRNA 18S inthe byabsence their assuggested rare, theyare or because C. neogracilis Chaetoceros Chaetoceros Brightwell strains belonging to Clade II, isolatedinthe CPH9 (Fig.strains belonging was toClade 3A), ). Similarly, reportsof thedoubtful tween Arctic strains of strainsof tween Arctic C. neogracilis species notisolatedin foundinthephytoplankton countswere Clade III

(http://malina.obs-vlfr.fr; see Bosak et al. 2015fora etal. Bosak (http://malina.obs-vlfr.fr; see C. neogracilis

strains were isolated from DCM waters. During the isolatedfromDCMwaters.Duringstrains were the strains from Clade I wellstrains fromCladeII asallthe as and Clade ce waters (Table 1), whereas 5 out of 8strainsof 5 1),whereas (Table ce waters show similarities with specimensfrom other with show similarities radiation, higher temperatures andlowernutrient higherradiation, temperatures to DCM waters. We do not know whether these these donotknowwhether to DCMwaters.We for these genotypes. However surface genotypes genotypes surface However genotypes. these for similar to those characterizing the Beaufort tothosecharacterizing Sea. the similar C. wighamii C. neogracilis C. neogracilis , as suggested by the morphologicaland , assuggested by the C. wighamii (see fig. 224 in Jensen &(see Moestrup inJensen fig. 224 described inthisstudy described have identical T-RFLP have identical from the BalticSea C. gelidus

and and C. (McNeill et al. 2012) to establish each of the four clades as valid species and toassessifone of clades speciesand (McNeillfour asvalid each ofthe etal.2012)toestablish fungi, and plants forCode ofNomenclatureInternational algae, revision required by the our study. has notbeenobserved in spore thatunfortunately (Schütt 1895)includesaspiny descriptionofthespeciesSarno, pers.observation). Theoriginal species considered solitary, as it has also been observed in the related species species species intherelated consideredalso beenobserved solitary, asithas abilitycolonies. Notably, tooccasiona the be associated with the seasonal ice and the shifts ice andthe with theseasonal be associated the Balticgenetic simila and theKattegat, Sea the and both Beaufort Sea the and salinity between intemperature Despite differences thesignificant etal.2012b). Balzano etal.2012a, (Balzano Baltic from the Sea or environmentalsequences Seaduring theMA Beaufort ofthe waters surface isolatedfrom flagellates the as wellphotosynthetic number sequences ofenvironmental Interestingly, a etal.2002). (Lundholm been isolatedfromKattegat 520, Fig. whichhas 2A) Balticand Sea, a solitary species whereas someoftheMALINA strains a solitary whereas species Indeed,formation. of colony absence with aprevalent described as Accepted cruise shareasimilarcellisolated during theMALINA morphology with (Rine worldwide collected and notrelatedtaxa todifferent thename mostprobably attributed has been cell-taxa, insuchsmall singlefeatures description andtothelackofdistinctive tothescantyspecies original (Schütt1895).Due Article Based onthe The Chaetoceros neogracilis species complex. Chaetoceros neogracilis Chaetoceros The Chaetoceros speciescomplex. neogracilis This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. Chaetoceros gracile Chaetoceros C. closterium C. closterium

available information, it is not possible to provide the authoritative taxonomic taxonomic information,itisnotpossible toprovidetheauthoritative available RCC1985 formsaclade, inthe28SrRNAtree,withastrain (K- Schütt fromtheBalticas solitary, Sea small lly form coloniesiscommontoother form lly s & Hargraves 1988). All the Arctic strains s &Hargraves 1988).AlltheArctic strains insalinity intheseenvironments. occurring rities found in samples from theseareasmight foundinsamplesfrom rities LINA cruise are genetically related tostrains genetically related LINA cruiseare C. neogracilis

have been observed forming observed short have been

was originally described as was described originally C. neogracilis was originally C. tenuissimus Chaetoceros Chaetoceros , together

(D. green alga Arctic Arctic green alga En Sea(Terradoetal.2013). Beaufort Bay andthe for suggested beenrecently genotype. Endemism has to anArctic correspond etal. 2007)andmight (Haitao complex thisspecies for lineage described meantime, we propose thattheArcticmeantime, wepropose material of compared withthetype informati additional ultrastructural them corresponds to molecular signatures described here asaffiliated are to molecular described considered signatures addition, theMALINA of strain by identified Arctic phylotypes their 18SrR arctica this study confinedtothe northern/polar area, including adistribution have Theprovisionalascriptionofthename complex. Acceptedas species, named here andbut genetically distinct(Figs.relatedcorresponds probably undescribed and7) toa 1,2B, this studyAnM0002, CC by thestrains Article as identified has been Antarctic frequently which species, watersBaltic of which isthetype inthe Sea, locality http://www.sccap.dk/) belongingII toClade of CPH9, syn issupportedby(i.e., K-1665, thestrains complex the factthatoneof Biogeography ofArcticdiatoms. (Percopo et al. 2016), andthe et al.2016), (Percopo This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. Micromonas Micromonas C. neogracilis C. neogracilis Chaetoceros (Lovejoy et al. 2007), several foraminiferan species (Darling etal. (Darling species several foraminiferan (Lovejoy etal.2007), C. closterium on on a larger number of strains from thefour tobe clades on alarger ofstrainsfrom number

C. neogracilis sensu stricto. Further analyses are required toprovide required are analyses sensu stricto.Further Most of the diatom species (10 out of 17) characterized in characterized Most ofthediatomspecies(10out17) sp. sp. C. neogracilis C. neogracilis Chaetoceros Chaetoceros MP187, CCMP189 andCCMP190(Choietal.2008), NA gene (Lovejoy & Potvin 2011) (Table 2). In& (Lovejoy Potvin2011)(Table2). gene NA (RCC1985)isphylogenetically distantfromany the species complex, wasthe species isolatedfrom complex, Danish C. neogracilis and eventually designate an epitype. In the In the anepitype. designate andeventually demic polar species include in particular the inparticularthe include demic polarspecies strains sharing very similar morphology and morphology strains sharing similar very species a number of Arctic protists fromtheBaffinof Arcticprotists anumber C. neogracilis C. neogracilis

complex, which was one of the few which was oneofthe complex, to

the Arctic C. neogracilis C. neogracilis . Themorphologically similar and is represented in isrepresented and Pseudo-nitzschia Chaetoceros species

Indeed, the northern/polar ecotype of the worldwide-considered species, species, the worldwide-considered ecotype of Indeed, thenorthern/polar isola genetically distinctandreproductively that populationspreviously are cosmopolitan tomake often thought species upunique or some example in widely asfor distributedmorphospecies, wide geographical distribution(Table 2). Molecu cold currents. belt across oceanconveyor theglobal globe orotherdeep the might via transported have been Such resting s formsal. 2003). could (Montresor et globe due across the or transport cells during their thesurvivalof at wouldhave lowlatitudes permitted colderseawater last glacial period,where the during occurred associated withamigration bipolar species couldbe of waters. Thepresence polar species isthusunlikely fromtran toarise hardly intemp can survive 2014). Polarspecies al. 2003)andtheciliate (Montresor et wellHasle asthe 2008)as and the Antarctic, has been suggestedfortwo and theAntarctic,hasbeen suchastheArctic environments, geographically distant but same species related inecologically the distribution (McMinnThe presence of et etal.2005,Whittaker al.2012,Goes etal.2014). Ehrenberg diatoms terrestrial 2007, Pawlowskietal.2008),andtheAntarctic

Accepted Skeletonema Article Few (5) of the strains characterized(5)Few inthisstudy ofthestrains supposed tohavea belongthatare tospecies Two species found here, Two species foundhere,

and This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. Hantzschia amphioxys Hantzschia (Kooistra Otherst etal.2008)species. Porosira glacialis (Ehrenberg) Grunow (Ehrenberg) Euplotes nobilii Polarella glacialis Polarella ted (Kooistra et al. 2008, Casteleyn et al. 2010). et al.2010). etal.2008, Casteleyn(Kooistra ted Fragilariopsis sport of living cells between Arctic and Antarctic Arcticand ofliving between cells sport and erate and tropical waters and theevolution of and erate and waters tropical lar methods have demonstrated conspecificity conspecificity methodshavedemonstrated lar to more recent transport of restingforms transport tomorerecent T. gravida urvive tropical waters or inalternative they or urvive tropicalwaters ValbonesiLuporini & udies on biogeography indicate indicate udies onplanktonbiogeography Pseudo-nitzschia Pseudo-nitzschia

Montresor, & Procaccini Stoecker (Souffreau et al. 2013). et al. (Souffreau Hustedt ,

are considered to havebipolar consideredare to Pinnularia borealis

species (Lundholm & C. socialis (Lelong etal.2012)

(Di Giuseppeetal.

,

has been

screening of environmental samples. environmental screening of patterns inprotistsusing largescalewill allowtotesttheoriesondispersalandbiogeographic forreference arctic diatomswillfaci sequences otherfarare species(Finlay & Fenchel2004), cosmopolitan species, as demonstr cosmopolitan species,as ofbipolar or populations geographical allow toidentify among markers would differences sensitive molecular thattheuseofmore cannotexclude We 2011). al. (Syvertsen 1977,Saret despite previous studiessuggesting thatthetwomo bipolar likely to al. 2012b),are tocorrespond (Balzano et theMALINA& cruise thosereportedfor 2002,Sukhanovaetal.2009),including Wassmann glacialis the28SrRNA genefound among sequences willbe Further analyses (Casteleyn etal.2010). Subsequently all the previous reportsof Subsequently alltheprevious Accepted Articleevidence and molecular species,i.e., as adistinct recently described Therefore while some species distribution patterns seem to support the hypothesis ofubiquity seem tosupportthehypothesis Therefore distributionpatterns whilesomespecies Similarly, the degree of interspecific divergence between the cosmopolitan the between Similarly, ofinterspecific divergence thedegree T. gravida .

This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. advocates they should be treated as separate species (Whittaker et asseparate species they al.2012), shouldbetreated advocates

(Degerlund et al. 2012, Huseby et al. 2012, Chamnansinp etal.2013). et al.2012,Chamnansinp al. 2012,Huseby (Degerlund et ated for the cosmopolitan species cosmopolitan ated forthe

C. socialis C. gelidus, of the Arctic and Antarctic strains of strains and Antarctic Arctic of the more restricted. The availability ofvalidatedThe availability more restricted. litate theinterpretation and ofmetabarcoding data required to evaluate the slight toevaluate here difference the required C. gelidus rphotypes are likely to be a single species species asingle arelikelyrphotypes tobe in Arctic waters (Booth etal.2002,Ratkova in Arcticwaters based on physiological, morphological morphological basedonphysiological, .

Pseudo-nitzschia pungens Pseudo-nitzschia T. rotula Porosira andthe

work, the ASSEMBLE EU FP7 research infrastr work, theASSEMBLE FP7 research EU particular ANR(ANR in the MALINA project, mainly supportedby We thankall help.Thisworkwas participantstoMALINA fortheir cruise Aizawa, R.W.2005.Living C.,Tanimoto,M.& Jordan, from diatomassemblages NorthPacific EMsupport. for SZN) Service, (Electron Microscopy Iamunno MaCuMBA R.Graziano grateful toF. (FP7-KBBE-2012-6-311975). are and We ANRProjectPhytIP fellowships,the andRS Balzano, S.,Gourvil,P.,Siano,R.,Chanoine,M., of cycle M. 2008.Morphology,Amato, A.&and sexual phylogeny, Montresor, L., T.& H. Amato, A.,Kooistra, M. Mann,D.G.,Proschold, H.C.F., Montresor, Ghiron,J. W. Jansen, phylogeneticR. K.& theRubicon: Alverson, A.J., Theriot,E.C.2007.Bridging Balzano, S., Marie, D., Gourvil, P. & Vaulot, D. 2012b. Composition ofthe P.&Gourvil, Vaulot, summer Balzano, S.,Marie,D., D.2012b. Balzano, S., Abs, E. & Leterme, S. C. 2015. Protist diversity along a salinity gradient in a coastal gradientBalzano, S.,Abs,E. &Leterme, S.C. 2015. Protistdiversity inacoastal asalinity along Booth, B. C. & Horner, R. A. 1997. on the Arctic Ocean Section, 1994: species 1994: theArctic Section, Ocean R. A.1997.Microalgae on &Booth, Horner, B.C. L.,Bérard-Therriault, M.& Bossé,L. Poulin, 1999. AcceptedBooth,Larouche,P., Belanger, B. C., S.,Klein,B.,Amiel,D.&Z. 2002.Dynamics Mei, of P. Article ACKNOWLEDGMENTS ACKNOWLEDGMENTS Oceanogr. summer1999. during waters and Bering Seasurface nitzschia mannii pseudodelicatissima Oceans insummer. andArctic inthenortheastPacific flagellates ofcultured photosynthetic 2012a. Diversity 158:193-207. among2007. Reproductiveisolation sympatric diatoms. of colonizations repeated analysis reveals J 18SrRNAgene RFLP ofthe andsequences nanoplankton communit photosynthetic picoand lagoon. abundance andbiomass. abundance Canadian etdugolfe du Saint-Lauren marin del'estuaire Oceanogr. Chaetoceros socialis 6:1480-98. 6:1480-98. This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. Aquat. Microb. Ecol. Aquat. Microb. Mol. Phylogenet.Evol. NRC Research Press, Ottawa, 387 pp. Press,NRC Research Ottawa, 387pp. 52:2186-205. 52:2186-205. 49:5003-25. 49:5003-25. sp. nov. (Bacillariophyceae): a pseudo-cryptic species within the within species sp.nov. a pseudo-cryptic (Bacillariophyceae): Biogeosciences complex. complex. blooms in the North Water. bloomsintheNorthWater. Deep-Sea Res. Part II-Top. Stud.Oceanogr. Res.PartII-Top. Deep-Sea 74:263-77. Phycologia 45:193-210. REFERENCES 9:4553-71. 9:4553-71. oPol (ANR-15-CE02-0007-02) and the EU project and theEUproject (ANR-15-CE02-0007-02) oPol -08-BLAN-0308), which funded SB post-doctoral SB funded post-doctoral which -08-BLAN-0308), ucture initiative(E ucture marine and fresh waters by thalassiosiroid thalassiosiroid fresh by waters marine and 47:487-97. Marie, D., Lessard, S., Sarno, D. &Lessard, S.,Sarno,D. Vaulot,Marie, D., D. from flow cytometry sortedsamples. flow from Guide d'identification Guide d'identification du phytoplancton t incluant protozoaires égalementcertains cryptic species in marine diatoms. inmarinediatoms. cryptic species ies in the Beaufort Sea assessed by T- ies intheBeaufort Seaassessed Deep-Sea Res. Part II-Top. Stud. Res.PartII-Top. Deep-Sea Deep-Sea Res. Part II-Top. Stud. Res.PartII-Top. Deep-Sea U-RI-227799) whichfunded U-RI-227799) 44:1607-22. 44:1607-22. Pseudo- ISME Protist P.

Casteleyn, G., Leliaert, F., Backeljau, T., Debeer, A.-E., Kotaki, Y., Rhodes, L.,Lundholm, Kotaki,Y.,Rhodes, N., A.-E., Leliaert,Casteleyn, T.,Debeer, G., F.,Backeljau, M. 2004.Seasonalcontributionsofphytoplankton andfecalCaron, G.,Gosselin, Michel,C.& Beaufort shelfofthe 2002.Oceanography& oftheCanadian R.W. MacDonald, Carmack, E.C. Brugel, Tremblay, S.,Nozais,C.,Poulin, M., J.-E Morphological studyBosak, M.&D. 2015. S.,Gligora Sarno, of Udovic, Chamnansinp, A., Li,Lundholm,Global diversityChamnansinp, A., oftwo O.2013. & Y., N. Moestrup, C Degerlund, M. & Eilertsen, H. C. 2010. Main species characteristics of phytoplankton spring ofphytoplankton Degerlund, characteristics & H. C. 2010.Mainspecies Eilertsen, M. Degerlund, M., Huseby, S., Zingone, A., Sarno, D. & Landfald, B. 2012. Functional diversity in Landfald, &Functional diversity Degerlund,S., Zingone,D. B.2012. A.,Sarno, M.,Huseby, reveals GlobalmolecularDarling,M. & phylogeography Kucera,K. F., C.M.2007. Wade, Crawford,L. R.M.,Gardner,C.& Thegenus Medlin, K.1994. D.,Gosselin,Coupel, P.,Matsuoka,A.,Ruiz-Pino, Lovejoy,& C.2011.Arctic Carmack, E.C. Li,K. W.,Tremblay,Comeau, A.M., J.-E., W. Is"biological ineukaryotes? Coleman, A.W.2009. the level of there species" key amolecular to Coleman, A. W. 2001. Biogeography and speciation inthe Coleman, A.W.2001.Biogeography andspeciation Cleve, P. 1896. Diatoms from Baffins Bay and Davis Strait. BayDavis Strait. Cleve, and P.1896.DiatomsfromBaffins H.M., Hong,Choi, H.G.,Joo, Jung, W., Morphology S.S.,Kang, S.& and Kang,2008. J. S.H.

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Di Giuseppe, G., Erra, F., Frontini, F. P., Dini, F., Vallesi, A. & Luporini, P. 2014. Improved Frontini,F., Vallesi, F.Luporini, P.2014. A.&Di Giuseppe,G.,Erra, P.,Dini,F., Hasle, G.R. 1964. Hällfors, ofBaltic G.2004.Checklist someheterotrophic Sea phytoplankton species(including Hall, T. A. 1999. BioEdit: a user-friendly biolog Hall, auser-friendly T.A.1999.BioEdit: V9 V4and Comparing & thehyper-variable Bunge, Stoeck, J. Dunthorn, M.,Klier,J., T.2012. Guo, L., Y.&Liu, Comparison ofpotentialdiatom'barcode'Guo, Z.,Zhang,S., Ren, Y.2015. Sui, there? are algaemany Guiry, of How species M.D.2012. microbial T. 2004.Cosmopolitan Finlay,Fenchel, &metapopulations offree-living B. J. 1985.Confidencelimitsonphylogenies.Felsenstein, Anapproachusing thebootstrap. J. Haitao, L.,Haitao,Ying, Guanpin,Z. Y.,Xiufang, S.,Suihan,W.& 2007. Guinder, V. A., Molinero, J. C., Popovich, C.A., J. E.&Guinder, V. A.,Molinero, Marcovecchio, Sommer,U. J. 2012. L.,LeGuillou, M.J., Romari,K.,Pedros- Gall, Chretiennot-Dinet, Eikrem, F., W., R., Massana, I., Chekalyuk, R.,Haugen, E.M.,McKee,K.T.,D'Sa,D. A.M., Goes, E.J., J. Gothes,H. Gu, H., Zeng, N., Xie, Z., Wang, D., Wang, W. & Yang, W. 2013. Morphology, phylogeny, and 2013.Morphology,Yang, W. Z., Wang, phylogeny, & Zeng, D.,Wang, W. Gu, H., N.,Xie, B. R.A.&Booth, C.1997.New Levasseur, P.A.,Horner, M., Wheeler, Gosselin, M., Accepted L. invertebrates. Guillard, R. Culture ofphytoplankton forfeeding marine 1975. Article description of the bipolar ciliate, description ofthebipolar protistan groups). program 95/98/NT. forWindows Euplotes Klasse. Nyserie utgitt avDet Norske Videnskaps-Akademi Lanceolate. group and Nitzschiaella I. the microscopes. species Somemarine of Eukaryot. Microbiol. diversity. of ciliateenvironmental assessment regions ofthesmallsubunitrDNAfor Journal of Ocean University ofChina Journal ofOceanUniversity gene ofrbcL andSSUrDNA. aswasevidenced by thevariations species complex Bahía Blanca Estuary, Argentina. and determining species taxonomyBacillariophyta. species and determining inthe ITS,gene and genes COI, (the18SrRNA rb . Evolution 65:1369-80. Dominance of the planktonic diatom Dominance oftheplanktonic marine ecosystems.fromcoastal oceanicand samples andnovelisolatesretrieved ofenvironmental nuclear SSUrDNA sequencing assessedby prasinophytes Alio, C.& picoplanktonic Diversity Vaulot, direct D.2004. of Publication Corporation, New York,pp.29-60. Stud. Oceanogr. Res.PartII-Top. Deep-Sea a ColdPoolduring of summer. competency inthepresence structure andphotosynthetic characterization pigment andmicroscopic P.J., Sait Stoecker, D. K.,Stabeno, Res. PartII-Top.Stud.Oceanogr. toxicity of Atama complex (Dinophyceae) from the ChukchiSea. from (Dinophyceae) the toxicitycomplex ofAtama al andice measurements ofphytoplankton L. & Chanley, M. H. [Eds.] L.& Chanley, M.H.[Eds.] This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. phylogenetic tree. phylogenetic 39:783-91. Nitzschia Protist 16:1-48. Baltic Sea Environment Proceedings Baltic SeaEnvironment 155:237-44. 59:185-87. and Fragilariopsis Eur. J.Protistol. Culture ofmarineinvertebrate animals. Euplotes petzi, Nucleic Acid symposium Series Nucleic Acidsymposium 44:1623-44. 44:1623-44. J. PlanktonRes. oh, S.& Sambrotto, R.N.2014.Fluorescence, Thalassiosira minima 6:167-74. ical sequence alignement editor and analysis andanalysis editor alignement sequence ical speciesand electron studiedinthelight iOslo.IMatematisk-Naturvidenskapelig gal production in the Arctic Ocean. intheArcticOcean. gal production of Bering Sea phytoplankton community phytoplankton of Bering Sea 109:84-99. 109:84-99. cL) and their effectiveness in discriminatingcL) and theireffectivenessin 50:402-11. Protist and definitionofitsbasal and positioninthe 34:995-1000. J. Phycol. 155:193-214. 95:1-208. Int. J. Syst. Evol. Micr. J.Syst. Evol. Int. in recent summers inthe inrecentsummers Cylindrotheca closterium 48:1057-63. 48:1057-63. 41:95-98. 41:95-98. Polar Biol PlenumBook In 36:427-36. Deep-Sea Deep-Sea : Smith, W. : Smith,W. Skrifter

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Arctic

Lee, M.-A., Faria, D. G., Han, M.-S., Lee, J. & Ki, J.-S. 2013.Evaluationofribosomal nuclear J.-S. &Lee, Ki, J. Han, M.-S., Faria, D.G., Lee, M.-A., Logares,L.,B Bass, D.,Bittner, R.,Audic,S., Logares, R.2012.Diversity C.& R.,Audic,S.,Santini,Pernice,M.C.,deVargas, Massana, inthe species Lim,Leaw,Lim, C.P.& 2013.Three novel H.C.,Teng, S.T., P. T. Soudant, P.&Lelong,A., Hégaret, Bates, H., S. 2012. S., Lundholm, N., Moestrup, O., Kotaki, Y., Hoef-Emden, K., Scholin, C. & Miller, P. 2006. Inter- P. 2006. K.,Scholin,C.& Miller, Lundholm, O.,Kotaki,Y.,Hoef-Emden, N.,Moestrup, & Bacle,Distribution vonQuillfeldt, J. L., Martineau, C. M.J., Legendre, H. 2002. Lovejoy, C., Kawachi,Lepère, M.,Romac,S C.,Demura,M., inevolution.A B. L., M.1989.Dinoflagellates Herzog, & Michot, Lenaers, G.,Maroteaux, Lovejoy, C. &Lovejoy, distributioninadynamic Potvin,M.2011.Microbialeukaryotic C. and Beaufort Sea R.,Potvin,M., Terrado, F.,Lovejoy, Bonilla, W. 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Nova

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Accepted Article in & picoeucaryotes D.2005.Mapping F.,Marie,D. Vaulot, Zhu, of F., R.,Not, Massana, 52:79-92. marine ecosystemsquantitative PCRofthe 18SrRNAgene. with This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. Fems Microbiol. Ecol. Fems Microbiol.

Table 1. Accepted Thalassiosiraceae Attheyaceae Fragilariaceae Article Bacillariaceae Family List isolatedduring theMALINA ofthestrain This article is protected by copyright. All rights reserved. AllrightsThis article isprotected by reserved. copyright. Porosira glacialis Porosira glacialis Porosira glacialis Thalassiosira nordenskioeldii Thalassiosira nordenskioeldii Thalassiosira nordenskioeldii Thalassiosira minima Thalassiosira minima Thalassiosira minima Thalassiosira Thalassiosira gravida Thalassiosira gravida Attheya septentrionalis Attheya septentrionalis Attheya septentrionalis Synedropsis hyperborea Synedropsis hyperborea arctica Pseudo-nitzschia arctica Pseudo-nitzschia arctica Pseudo-nitzschia arctica Pseudo-nitzschia Pseudo-nitzschia Pseudo-nitzschia Pseudo-nitzschia pellucida Nitzschia Cylindrotheca closterium Species cf. hispida hispida

granii granii granii RCC2273 RCC2008 RCC2006 RCC2276 RCC1985 Strain code RCC2039 RCC1995 RCC2522 RCC2021 RCC2000 RCC2269 RCC2266 RCC2265 RCC2521 RCC1999 RCC1984 RCC2042 RCC1988 RCC1986 RCC2520 RCC2043 RCC2517 RCC2005 RCC2004 RCC2002 cruise and used in the present study. the presentstudy. cruiseandusedin 1 PAC060709A PAC080709A PAC080709A BEA130709A 280 Station 690 690 620 680 690 PAC050709A 394 394 680 280 280 680 280 280 280 280 690 690 690 690 Isolation site Isolation

(m) Depth 2 30 29 29 29 40 30 30 30 30 30 30 29 29 29 29 0 5 5 0 3 3 0 3 3 3 LM, TEM, SEM SEM TEM, LM, SEM TEM, LM, SEM TEM, LM, SEM TEM, LM, SEM TEM, SEM TEM, LM, LM LM LM SEM TEM, LM, LM SEM TEM, LM, SEM TEM, LM, TEM LM, SEM TEM, LM, SEM LM, TEM, LM TEM LM, LM,TEM TEM LM, Morphology LM LM TEM SEM, LM, LM SEM TEM, LM, 3 JN934676 JN934691 JN934669 JN934675 JN934671 numbers Genbank accession JN934673 JF794045 JF794040 JF794051 JF794046 JF794052 JF794039 18S JQ995415 JQ995444 JQ995441 JQ995440 JQ995464 JQ995414 JQ995402 JQ995433 JQ995405 JQ995404 JQ995463 JQ995434 JQ995461 JQ995419 JQ995418 JQ995416 JQ995391 JQ995421 JQ995420 JQ995450 JQ995403 JQ995432 JQ995428 28S ITS 4 Accepted Article Chaetoceraceae Hemiaulaceae Cymatosiraceae This article is protected by copyright. All rights reserved. AllrightsThis article isprotected by reserved. copyright. Chaetoceros neogracilis Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros gelidus Chaetoceros gelidus Chaetoceros gelidus Chaetoceros gelidus Chaetoceros gelidus Chaetoceros gelidus Chaetoceros gelidus Chaetoceros gelidus Chaetoceros decipiens Chaetoceros groenlandica Eucampia groenlandica Eucampia groenlandica Eucampia cornucervis Arcocellulus Shionodiscus bioculatus

neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis clade I clade I clade I clade I clade I clade I clade I clade I clade I clade I clade I clade I clade I clade I clade I clade I clade clade I clade

RCC2271 RCC2046 RCC1994 RCC1992 RCC1990 RCC1997 RCC2038 RCC2037 RCC1996 RCC2270 RCC1991 MALINA S509 MALINA S509 MALINA S502 P27.B3 MALINA S441 P21-E6 RCC2507 RCC2281 RCC2280 RCC2279 RCC2278 RCC2275 RCC2274 RCC2267 RCC2264 RCC2263 RCC2262 RCC2017 RCC2011 RCC2003 MALINA S135 MALINA E65PG18 MALINA E65PG4 690 280 690 620 620 690 690 690 690 ARC120709A 620 760 760 320 235 760 760 320 320 620 620 394 235 235 460 760 620 690 BEA140709A 690 690 30 29 65 65 29 29 29 29 65 25 29 29 29 3 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 0 LM LM LM LM LM LM LM LM LM LM TEM LM, LM LM LM,SEM LM SEM LM, SEM TEM, LM, LM SEM TEM, LM, SEM LM, SEM LM, SEM TEM, LM, SEM,TEM LM, SEM TEM, LM, LM LM JN934677 JF794042 JF794044 JF794043 JF794041 KT884482 JQ995455 JQ995454 JQ995453 JQ995452 JQ995449 JQ995448 JQ995442 JQ995439 JQ995438 JQ995437 JQ995427 JQ995423 JQ995417 JQ995396 JQ995393 JQ995446 JQ995435 JQ995411 JQ995409 JQ995407 JQ995413 JQ995431 JQ995430 JQ995412 JQ995445 JQ995408 JQ995399 JQ995397 JQ995459 KT860532 KT860531 KT860530 KT860529 KT860527 KT860526 KT860523 KT860522 KT860521 KT860520 KT860517 KT860513 KT860511 KT884482 KT860540 KT860541 KT860536 4 3 2 1 Electron Microscopy.

without an RCC code are no longer available. without anRCCcodeare nolonger RCC:Roscoff culture collection. More Please notethat the V4 region of the 18S rRNA gene has beensequenced from all thestrains. Technique usedfor the morphological identification: LM, Light Mi Accepted Samplinglocation of Article This article is protected by copyright. All rights reserved. AllrightsThis article isprotected by reserved. copyright. Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros neogracilis Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros Chaetoceros neogracilis Chaetoceros the MALINA cruise. See Table S1for more details

neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis neogracilis information on thestrainshttp://roscoff-culture-collection.org/ isavailable at clade IV clade IV clade IV clade IV clade IV clade IV clade cladeIII cladeIII II clade II clade II clade II clade II clade II clade II clade I clade I clade clade II clade I clade

MALINA E43.N2 MALINA E43.N2 RCC2506 RCC2318 RCC2282 RCC2277 RCC2272 RCC2268 RCC2261 MALINA S512 MALINA S511 MALINA S510 MALINA FT56.6 PG6 RCC2022 RCC2016 RCC2014 RCC2012 RCC2010 RCC1993 RCC1989 croscopy; TEM, Transmission Electron Microscopy; SEM, Scanning

BEA140709A 235 620 760 BEA130709A BEA130709A BEA130709A 460 760 760 760 110 680 760 110 110 620 620 620 65 65 65 0 3 3 0 0 0 3 3 3 3 3 3 3 3 3 3 LM, SEM SEM LM, SEM LM, TEM LM, SEM,TEM LM, SEM LM, LM LM LM LM LM LM LM,SEM LM LM JN934684 JF794049 . Strains KT884485 KT884484 KT884483 JQ995395 JQ995429 JQ995426 JQ995425 JQ995424 JQ995422 JQ995410 JQ995406 JQ995392 JQ995458 JQ995457 JQ995456 JQ995451 JQ995447 JQ995443 JQ995436

KT860542 KT860518 KT860516 KT860515 KT860514 KT860512 KT860510 KT860509 KT860535 KT860534 KT860533 KT860528 KT860525 KT860524 KT860519 KT884485 KT884484 KT884483 (Østrup) Crawford (Østrup) Crawford Attheya septentrionalis & Syvertsen (Grunow) Hasle, Medlin Synedropsis hyperborea Percopo & Sarno Pseudo-nitzschia arctica (Hasle) Hasle Pseudo-nitzschia granii Grunow Nitzschia pellucida Reimann Lewin(Ehrenberg) & Cylindrotheca closterium Species Accepted Article Table 2.

Geographic distributionandmorphological references This article is protected by copyright. All rights reserved. AllrightsThis article isprotected by reserved. copyright.

Stonik etal. 2006 et al.1994 Crawford Hasle etal.1994 Percopo etal.2016 Marchetti etal.2008 references therein and Hasle 1997, & Syvertsen therein and references Bérard-Therriault et al. 1999, &Jahn Kusber2005 references therein and Hasle 1997, & Syvertsen references Morphological

study 2012) Common inArcticwaters Cosmopolitan (Hasle &Syvertsen 1997) Global distribution (Hasle &(Hasle Syvertsen 1997) totemperateNorthernregion cold water 1994) Common in Arctic waters Syvertsen 1997) &(Hasle region Northern coldwater al. 2016) (Percopo et waters Arctic from Recently described Subarctic Pacific Northern Atlantic &(Hasle Syvertsen 1997) totemperate? Northernregion coldwater 2002) Ishigaki Island, Japan environments European freshwater Antarctica Therriault etal.1999) (Bérard- region Northern coldwater of the species identified in the present study. study. identifiedinthepresent of thespecies )

(Hällfors 2004) (Hällfors

(Marchetti etal. (Marchetti (Hasle 1964) (Hasle

(Lundholm etal. (Lundholm

(Hasle etal. 2008, this (C ă r ă

us Sea Frobisher Bay,BarentsGreenland, Bayet al.2016) (Percopo BarrowBeaufort Baffin Strait, Sea, et al.2016) andBarents (Luddington White Seas 2003) Chukchi Sea(vonQuillfeldt etal. 1964) Norwegian (Hasle Sea Chukchi Sea(vonQuillfeldt 2000) 2009) Central Arctic (Katsukietal. Ocean al.2000) et Laptev Sea(Tuschling al. 2016) (Luddington et andBarents Sea White Chukchi Sea(vonQuillfeldt 2000) 1982) Schrader & (Horner Beaufort Sea Distribution inArctic waters Chukchi Sea (vonQuillfeldt etal. NansenBasin et al.1997) (Gosselin 2003) Chukchi Sea (vonQuillfeldt etal. (Hasle et al. 1994) et al.1994) (Hasle

Acceptednordenskioeldii Thalassiosira Gaarder Thalassiosira minima Syvertsen ArticleThalassiosira Cleve Thalassiosira gravida

This article is protected by copyright. All rights reserved. AllrightsThis article isprotected by reserved. copyright. cf. Cleve hispida

references therein and Hasle 1997, & Syvertsen al. 2007 Hoppenrath et references therein and Hasle 1997, & Syvertsen Syvertsen 1986 Syvertsen 1977

(Hasle &(Hasle Syvertsen 1997) totemperate region Northern coldwater Common inArcticwaters (Whittaker et al.2012) waterregions cold Northern andsouthern Arctic watersCommon in Common inArcticwaters &(Hasle Syvertsen 1997) totemperateNorthernregion coldwater systems et (Guinder al.2012) Warm waters incoastal 2016, as North AtlanticOcean (Luddington etal. al.2007) et North Sea(Hoppenrath &(Hasle Syvertsen 1997) Cosmopolitan excluding polar regions T. aff. minima

) andestuarine

Canadian Arctic (Aizawa etal.2005) (Aizawa Canadian Arctic 2016) Amundsen Gulf(Luddington etal. First reportinthisstudy 2003) Chukchi Sea(vonQuillfeldt etal. Quillfeldt 2000) (von Sea Barents Svalbard andthe 2009) Central Arctic (Katsukietal. Ocean 2016) Amundsen Gulf(Luddington etal. 2009) Central Arctic (Katsukietal. Ocean al. 2000) et Laptev Sea(Tuschling Baffin Bay et al.1997) NansenBasin, Chukchi Sea(Gosselin al.2000) et Laptev Sea(Tuschling al. 2016) (Luddington et andBarents Sea White Baffin Bay 2003) Laptev Sea (Tuschling et al.2000) et Laptev Sea(Tuschling 2010) &Barents (Degerlund Eilertsen Sea Baffin Bay

(Caron et al. 2004) et al. (Caron

(Lovejoy et al. 2002) et al. (Lovejoy (Caron et al. 2004)

AcceptedCleve Chaetoceros decipiens Cleve Eucampia groenlandica Syvertsen Hasle, vonStosch& cornucervis Arcocellulus KangTheriot & (Grunow) Shionodiscus bioculatus Article(Grunow) Jørgensen Porosira glacialis

Alverson, This article is protected by copyright. All rights reserved. AllrightsThis article isprotected by reserved. copyright.

and references therein and references Jensen & Moestrup 1998 &Jensen Moestrup1998 references therein and Hasle 1997, & Syvertsen &Syvertsen 1983 Hasle Hasle etal.1983 Bérard as references therein and Hasle 1997, & Syvertsen references therein and Hasle 1997, & Syvertsen Thalassiosira bioculata - Therriault et al. 1999, etal.1999, Therriault

: Common inArcticwaters Syvertsen 1997) region southern cold water totemperate,Northernregion coldwater Common in Arctic waters Cosmopolitan (Hasle &Syvertsen 1997) Common inArctic waters Syvertsen 1997) &(Hasle region Northern coldwater (Percopo etal.2011) Mediterranean Sea Zealand New and temperateNorthern waters, cold Common inArcticwaters Syvertsen 1997) &(Hasle region Northern coldwater (Hasle & 1997) Syvertsen (Hasle

(Hasle & 2002) 2009) al. 2016) Barents Sea (Luddington etal.2016) Barents (Luddington Sea al.2000) et Laptev Sea (Tuschling Baffin Bay (Cleve 1896) 2002) et al. Baffin Bay (Lovejoy 2009) Central Arctic (Katsukietal. Ocean Baffin Bay & Eilertsen2010) Norwegian coastal (Degerlundwaters White 2003) Chukchi Sea(vonQuillfeldt etal. 2016) Amundsen Gulf(Luddington etal. 2009) Central Arctic (Katsukietal. Ocean Seas andBarents White etal.2009) (Sukhanova Beaufort Sea al.1997) Chukchi Sea(Gosselin et 2016) Amundsen Gulf(Luddington etal. Central Arctic Ocean 2003) Chukchi Sea(vonQuillfeldt etal. Barents Sea (Ratkova & Wassmann Wassmann & (Ratkova Barents Sea Baffin Bay et al.2005) Sea(Aizawa Bering North Pacific and

and Barents Sea and Barents Sea

(Booth et al. 2002) (Booth etal. (Caron et al. 2004)

(Katsuki (Luddington et

(Olli etal. (Olli

et al.

Accepted Article (Schütt) VanLandingham Chaetoceros neogracilis Lundholm & Moestrup Chamnansinp, Li, Chaetoceros gelidus This article is protected by copyright. All rights reserved. AllrightsThis article isprotected by reserved. copyright.

Schütt 1895(as Chamnansinp etal.2013 See discussion

C. gracile ) 2012) et al. Baltic(Hällfors Majaneva Sea 2004, et al.2013) (Chamnansinp region Northern coldwater

2011) Sea Beaufort Svalbard (Choietal.2008) etal.2013) (Chamnansinp Greenland Barents NorwegianSea, Sea, & Eilertsen2010) Norwegian coastal (Degerlundwaters 2002)

(Lovejoy & Potvin

Ocean. Pacific indicate from strains 1. Asterisks inFig. theNorth isolated as described to thebranch nodes valuesare Likelihood joining next indicated Maximum andNeighbour andNJ (right). (left) ML associated of treesinwhichthe evolutionary historieswere i underlined. The whereas inbold labelled are sequenced here ( Figure 2. Ocean. Pacific isolatedfrom indicatewere theNorth strains corresponding for Asterisks the obtained node. (Neighbour- toright left (ML) tothebranchesfrom shownnext are in thebootstraptest(1,000replicates) replicate of trees the strain percentage code. The are Th waters isolated fromArctic underlined. otherstrains labelled inboldwhereas are here sequenced strains MALINA outgroup. The Corethron pennatum from centrics( isolated during radial cruise. Four sequences theMALINA strains thediatom foundwithin genotype each from one sequence atleast The treeincludes Figure 1. FIGURE LEGEND AcceptedA) Article pennate and ( pennate Full treederive 18SrRNAphylogenetic 28S rRNA phylogenetic tree inferred by treeinferred 28S rRNAphylogenetic This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. joining). Missing percentage values and “_” indicate that bootstrap values < 50 % bootstrapvalues<50 “_”indicatethat and values percentage Missing joining). B ) centric diatoms isolated during the MALINA cruise. The MALINA strains MALINA diatomsisolatedduring strains ) centric cruise. The theMALINA , Rhizosolenia setigera, taxa cluster together is shown next tothebranches on based isshownnext clustertogether taxa other strains isolated from Arctic waters are waters strains isolatedfrom Arctic other nferred using maximum likelihood. The percentage nferred using likelihood. maximum The percentage and e Genbank accession number is indicated to next number Genbank accession e in which the associated taxa clustered together taxa whichtheassociated in Rhizosolenia similoides d from Maximum Likelihoodd from (ML) analysis. Maximum maximum likelihood(ML)maximum forthe analysis ) have usedas been Corethron hystrix , bar, 0.2 rimoportula.Scale fieldand micrograph, viewof Noteapical RCC2043.External theapex. slit view of the apex. Noteapical slitfiel view oftheapex. Scalebar,0.1 apical slitfield andrimoportula. bar,1 of thevalve. Scale 2 in valveview.Scalebar, Scale bar, interruption. 2 which isvisibletheraphe Figure 4. 1. Figure as for tothebranches indicatednext values are of IT forthe treewhereas to rootthephylogenetic Chaetoceros neogracilis Figure 3. interspace. Scale bar, 1 bar, interspace. Scale bar, 20 nitzschia 1 Scale bar, poroids. RCC2006. Detail complete ofthevalvewithscattered Scalebar,0.5 poroids(arrows). fewthe valve. incomplete Notethe micrograph, RCC2006.Wholevalve. Scale bar,5 Scalebar,2 RCC2276. Detail ofcellapex. bar, 2 Scale larger interspace. central of thevalve. Notethe micrograph, RCC2276.Wholevalve. Scale bar,5

Accepted ArticleChaetoceros

μ μ

m. (I) TEMmicrograph, ofthecentral RCC2004.Detail valve.Note larger the arctica (A) m. 28S rRNA ( 28S rRNA This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. Cylindrotheca closterium sp. (CCMP187, CCMP189, sp. (CCMP187,CCMP189, CCMP190) bootstrap were The as outgroup. used : (H) LM micrograph, RCC2002. A colonyLM micrograph, Scale twocellsingirdle RCC2002. : (H) view. of A μ ), ITS-1), ( strains isolated from the Beaufort Sea. For the 28S, For the28S, Beaufort Sea. strainsisolatedfrom the m. (J-N) (J-N) m. μ μ m. (L) SEM micrograph, RCC2043. Internal view of the apex. Note m. (L) RCC2043. Internal SEM micrograph, oftheapex. view m. (K)SEMmicrograph, RCC2043.External viewof thecentral part B Synedropsis hyperborea ), and ( 5.8S+ITS-2 d andabsenceofrimoportula. Scale 0.5 bar, : TEMmicrograph, ofthe RCC1985.Detail valvein μ m. (E-G) m. (E-G) μ S-1 and 5.8S + ITS-2 trees, the Antarctic strains ITS-2 trees,theAntarcticstrains and5.8S + S-1 m. (M) SEM micrograph, RCC2043.External m. (M)SEMmicrograph, μ μ μ m. (F) TEMmicrograph, RCC2006.Detail of m. (C)TEMmicrograph, RCC2276.Detail m. (B-D) Pseudo-nitzschia granii Pseudo-nitzschia C ) phylogenetic treesforthestrains of ) phylogenetic : (J) LM : (J) RCC2043.Cell micrograph, μ m. (D) TEM micrograph, TEM m. (D) Nitzschia μ m. (G)TEMmicrograph,

pellucida μ C. gelidus m. (H-I) : (E) TEM : (B) TEM μ m. (N)SEM Pseudo- was used Figure 5. Accepted bar,1 Scale ofthevalve. face protrusions ontheexternal labiate-shaped thesmallexternal RCC2266. Detail oftwomarginal fultoportulaewith micrograph, RCC2266.FivecentralScale bar,fultoportulae. 1 Internal bar,1 viewofavalve.Scale (N) SEMmicrograph, RCC2269. therimoportulabe Note central fultoportulae. micrograph, viewof RCC2266.External with aring thevalve marginal of fultoportulae andtwo micrograph, chloroplasts.Scalebar,2 RCC2269.Cellingirdle view withtwo RCC2521. Detail ofthefultoportula.Scalebar, 0.2 Scale bar,1 and openbands. acomposed by thevalvocopula, copula bar,1 Scale foramina. and intheareolae margin minutespinesaremicrograph, presentonthehyaline shortand RCC2521.Detail ofa valve; Article bar,1 Scale marginal fultoportulae. betweentwo fultoportula. Notetherimoportula micrograph, RCC2521.Acellwith aring marginal invalveviewcentral fultoportulae of andone valveface scattered onthe micrograph,clus RCC1999.Avalvewith thecentral bar,20 joinedincolony.Three cells Scale (F) LM micrograph, RCC1999. RCC1986. AhornwithfourlongitudinalScale bar,0.5 strips. strips.Scalebar, 0.5 RCC2042. Ahornwiththree longitudinal TEM micrograph, RCC1986.Acircular bar, valve. 1 Scale Scale bar, 10 (A-E) (A-E) This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. μ m. (B) LM micrograph, RCC2042.Acell m. (B) Scale bar, view. 20 ingirdle Attheya septentrionalis . Scale bar,5 : (A) LM: (A) micrograph, RCC1986.Cellsingirdle view. μ m. (H-K) (H-K) m. tween twomarginal fultoportulae. 1 tween Scalebar, μ m. (J) SEMmicrograph,RCC2521. Them. (J) girdle Thalassiosira μ ter ofand several fultoportulae fultoportulae m. (L-P)m. μ m. m. μ m. (D) TEM micrograph, TEM m. (D) Thalassiosira minima μ μ μ m. (F-G) m. (F-G) m. (P) SEM micrograph, m. (P)SEM m. (E) TEM micrograph, m. (E)TEM cf. hispida μ m. (K)TEMmicrograph, μ m. (O) TEM m. Thalassiosira gravida μ : (H)SEM m. (G) TEM m. μ m. (M)SEM μ : (L) LM: (L) m. (I) TEM μ m. (C) μ m. : LM micrograph, RCC1997. Part of a colony. Scale bar,20 Scale Part ofacolony. LM RCC1997. micrograph, valve with the central rimoportula (arrow). Scale bar,1 Scale (arrow). rimoportula valve withthecentral colony.micrograph, RCC2037.Partofa Scalebar,5 Scalebar,10 marginal rimoportula(arrow). thevalve over numerous fultoportulaescattered RCC1995. Cellinvalve view. Scalebar,10 Acceptedthe twoocelluli are bar, Scale 1 visible(arrows). Figure 7. bar,2 Scale the pilusbase(arrows). bar,the ocelluli Scale 1 (arrows). bar, 1 pili (arrow). Scale ofthe branches conspicuous Notethe girdle view. cell in A slightlycurved valve.10 Whole Scalebar, 10 rimoportula.Scalebar, a subcentral valve centre and notethemarginal ring viewofacell; External Articlemicrograph, RCC1991.Acell view. ingirdle Scale bar, 20 LM RCC1995.Cell micrograph, Scale bar, 1 collar. bearing aterminal with long tubes external 2 fultoportulae. Scalebar, positioned withintwomarginal fultoportulaand onerimoportula fultoportulae, onecentral girdle Scale 5 bar, view. Figure 6. μ m. (K) SEMmicrograph, valve (right). RCC2270.Apili Note valve(left)and aprocess (A) (A-C) This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. Arcocellulus cornucervis Arcocellulus Thalassiosira nordenskioeldii μ m. (B) RCC2000.Avalve withamarginal TEMmicrograph, of ring μ μ m. (C)SEMmicrograph, RCC2000.Acell withringfultoportulae of m. (J-L) in girdle view. Scale bar,10 in girdle view.Scale μ m. (L) A pili valve in which the short spinules are visible near near are visible m. (L) inwhichtheshortspinules A pilivalve μ m. Arcocellulus cornucervis Arcocellulus : TEMmicrograph,which valvein RCC2270.Aprocess μ μ m. (G-I) m. (F) TEMmicrograph,RCC1995.Avalve with of fultoportulae, the single fultoportula inthe singlefultoportula fultoportulae, the of : (A) LM: (A) micrograph, RCC2000.Acolony in surface. Note the central annulus andthe annulus central Notethe surface. μ m. (B-C) μ Shionodiscus bioculatus m (C) SEM micrograph,m (C) RCC2037.A μ μ m. (D-I) m. (I)m. TEMmicrograph, RCC1991. μ μ m. (E) LMm. (E) RCC1997.A micrograph, Eucampia groenlandica Eucampia m. (H) SEM micrograph, RCC1991. m. (H)SEM μ μ : (J) SEMmicrograph,: (J) RCC2270. m. (E) LMmicrograph, (E) m. m. (D-F) (D-F) m. Chaetoceros decipiens Porosira glacialis Porosira : (G) LM: (G) : (B)LM : (D) : (D) : (D) micrograph, parallel RCC1997.Agirdleband with micrograph, RCC1997.Aterminalvalve.No Scale bar, 2 Scale bar, 2 central slit-likeprocess. (A-C) Figure 8. Scalebar, 1 aperture. with thenarrow spherical 50 bar, colony. Scale 20 thepicture.Scalebar, setae ontheupperpart of L) μ solitary Scalebar,20 cell. micrograph, RCC2271.Setae witharrowhe 2 the external tube.Scalebar, apertures. Scalebar,1 quite narrow bar,5 cells. Scale colony four of micrograph, RCC2016.Asolitary 10 cell. Scalebar, Scale bar, 10 Scale bar, 1 long Scale 5 bar, seta. ofthe spines inalarge part andthestraight long picture) upper part ofthe bar, 1

Accepted Article RCC1997.Two m. (G)SEMmicrograph, Chaetoceros gelidus Chaetoceros μ m. (B) SEMmicrograph, twotypes theshort(on ofsetae, RCC2271.Detail ofthe This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. μ μ μ m. (L) micrograph,Intercalary bar,2 TEM RCC2271, Scale valve. m. (D-N) (D-N) m. m. (E) LM micrograph,m. (E) RCC2017.Asolitary 10 cell. Scalebar, Chaetoceros gelidus Chaetoceros : (J) LM thetwostraight chain. Note (J) A curved micrograph,: RCC2271. Chaetoceros μ m. (F) TEM micrograph, RCC1997.Aterminalvalve.Scalebar,5 m. (F) TEMmicrograph, μ μ m. (J) TEMmicrograph,m. (J) RCC2012.Terminal valvewiththe m. (L) SEMmicrograph, RCC2271.Twointercalary valves μ m. (H)SEMmicrograph, ofa RCC2012.Detail Note colony. μ

neogracilis μ : (A) TEM micrograph, RCC2271. Intercalary valve. Scale Intercalary RCC2271. : (A)TEMmicrograph, valve. m. (I)micrograph, RCC2012.Terminal SEM valvewith μ m. (K) TEM micrograph,Intercalary TEM RCC2012. m. (K) valve. m. m. ad-shaped spines.Scalebar,1 ad-shaped intercalary bar,1 valves.Scale te the central process. Scale bar, 5 process.Scalebar, central te the seta (crossing the picture). Note theabsence of thepicture). (crossing seta : (D) LM: (D) micrograph, RCC2272.Asolitarycell. μ costae and small poroids. Scale bar, 5 poroids. costae andsmall μ m. (C)SEMmicrograph, RCC2271.Aspore. m. (K) LMm. (K) RCC2271.A micrograph, μ m. (G) LMm. (G) micrograph, RCC1989.A μ μ m. (N) SEM m. (H)TEM μ μ m. (F)LM m. (I) TEM μ m. (M)SEM μ m. (J- culture collections.CCMP: trees.Moststrains are phylogenetic study forthe aregenotypes inred. indicated thedifferent changes between neogracilis (www6.inra.fr/carrtel-collection_eng), CS:Au (www6.inra.fr/carrtel-collection_eng), of AlgaeandProtozoa(www.ccap.a Te Culture CollectionofAlgae atUniversityof InstitNIOZ: CultureCollectionatNetherland Collection at Culture UNC: (ncma.bigelow.org), Table S2. at RoscoffMost strainsareavailable CultureCollection(RCC) culture-collection.org). NaplesZoologica (www.szn.it),RCC:Anton Dohrn, Roscoff CultureCollection(http://roscoff- (www.csiro.au/en/Research/Collections/ANA neogracilis Figure 9. 0.5 bar, costae. Scale transverse by short interconnected micrograph, spiral RCC2271.Detail withspinesandlong costae ofaseta Accepted ArticleTable S1. Supporting information Diagrams of the secondary structure of the ITS-2 transcripts of( transcripts ITS-2 ofthe secondaryDiagrams structure ofthe List andspeciesfromwhich ofthestrains Details of the strain isolated during the MALI Details ofthestrainisolatedduring the Clade III Clade and( RCC1989, This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. I Clade RCC2279,( National CentreforMari B μ ) m. m. Chaetoceros neogracilis c.uk), TCC:ThononCultureCollection D ) Chaetoceros neogracilis Chaetoceros ute for Sea Research (www.nioz.nl), UTEX: (www.nioz.nl), ute forSeaResearch CC/About-our-collection), SZN:CC/About-our-collection), Stazione stralia National Alg xas Austin (utex.org/), CCAP:CultureCollection xas (www.unc.edu/), NorthUniversity Carolina of currently available at different available currently institutionsor ne Algae and Microbiota andMicrobiota ne Algae the sequences were used inthepresent used were the sequences NA cruise and used inthepresent study.NA cruiseandused Clade II Clade RCC2318,( Clade IV Clade RCC2010.Base ae Culture Collection ae Culture A ) Chaetoceros C ) Chaetoceros Chaetoceros present study. present study. Accepted Article Figure 6. asfor tothebranches bootstrapvaluesarenext indicated The were asoutgroup. used Figure S1. This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright. Phylogenetic tree of the ITSthe operonof tree ofthe Phylogenetic

The Antarctic strainsof The Antarctic Chaetoceros Chaetoceros sp. (CCMP187,CCMP189, CCMP190) Chaetoceros sp. strains isolatedinthe sp. strains Accepted Article This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright.

Accepted Article This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright.

Accepted Article This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright.

Accepted Article This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright.

Accepted Article This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright.

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Accepted Article This article is protected by copyright. All rights reserved. AllrightsThis articleisprotectedby reserved. copyright.

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RCC2281 Chaetoceros neogracilis MALINA S441P21E6 Chaetoceros neogracilis RCC2280 Chaetoceros neogracilis RCC2279 Chaetoceros neogracilis RCC2278 Chaetoceros neogracilis RCC2275 Chaetoceros neogracilis

44 RCC2274 Chaetoceros neogracilis RCC2267 Chaetoceros neogracilis Clade I RCC2264 Chaetoceros neogracilis RCC2263 Chaetoceros neogracilis 45 RCC2262 Chaetoceros neogracilis RCC2017 Chaetoceros neogracilis

82 RCC2011 Chaetoceros neogracilis RCC2003 Chaetoceros neogracilis RCC2507 Chaetoceros neogracilis MALINA S502P27B3 Chaetoceros neogracilis 45 RCC2016 Chaetoceros neogracilis 66 RCC2022 Chaetoceros neogracilis RCC2010 Chaetoceros neogracilis RCC2012 Chaetoceros neogracilis Clade IV 97 49 RCC2014 Chaetoceros neogracilis MALINA FT56.6PG6 Chaetoceros neogracilis RCC2261 Chaetoceros neogracilis RCC2268 Chaetoceros neogracilis RCC2272 Chaetoceros neogracilis RCC2277 Chaetoceros neogracilis 58 Clade II RCC2282 Chaetoceros neogracilis RCC2318 Chaetoceros neogracilis RCC2506 Chaetoceros neogracilis RCC1989 Chaetoceros neogracilis 99 RCC1993 Chaetoceros neogracilis Clade III CCMP190 Chaetoceros sp. CCMP189 Chaetoceros sp. 100 Antarctic Chaetoceros sp. CCMP187 Chaetoceros sp. 5 4VQQMFNFOUBSZ5BCMF4

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