Accepted Article vegetative cells enrobed by an ectoplasmic net; thraustochytrids that form a unilateral non- groups taxa:of morphologically-defined thelabyrinthulids thatform spindle-shaped (Bennett al. et 2017).Historically, haveLabyrinthulea been thedivided distinct intothree an ectoplasmic net (Tsui al.et 2009)that usedis tointerface nutrient acquisition and motility morphologically distinct heterotrophic unicellular other eukaryotesfrom ofthe by formation hypotheses consistently grouped our unknownisolate and closest affinity to nucleotide extraction, databasequeries of the reproduction bybiflagellate zoospore release,as well asbinarydivision. After and culturing Norway.Tromsø, Lightmicroscopyrevealed theof ectoplasmic production nets and from seawater pollen-baited acquired from nearshore the environmentproximityinto ABSTRACT This by is protectedarticle reserved. Allrights copyright. doi: 10.1111/jeu.12494 lead to differences between versionthis and the throughbeen the copyediting, pagination typesetting, which process, may and proofreading hasThis article been accepted publicationfor andundergone peerfull but review not has exceed 10 Labyrinthuleatheimportantdecomposers are ofthe detrituswhose pooland abundance can saprotrophs,bacteriovores, symbiontsand in THE Labyrinthulea(=Labyrinthulomycota Keywords haliotidis aplanochytrids, weerect anov.that gen. novel circumscribes our isolate andthe former describe our unknown isolate as a novel species. To resolve molecular polyphyly within the acquired from microscopy,electron information,history lifetesting, phylogenetic weand prolongedrevealedinteractions with hyphal strands. Basedthe oninformation combined bothrosomes. Co-culturing with experiments the fungusmarine walls ofcomprised electron-dense lamella that formed protuberances, some associated with homoplasious thraustochytrids. Transmission +4777645711;Telephone: Fax:+ Framstredet 6,Tromsø, 9019 Norway Corresponding author a UiTNorgesuniverstiet, arktiske NFHbygget, BFE, Framstredet 6, 9019 NorwayTromsø, SpeciesNew ofSaprobic ofThraustochytrid New Species Emendment and &Gardinger---Aplanochytrid Hassett Article :Original type Article DR. BRANDON THASSETT (Orcid 0000-0002-1715-3770) : ID BrandonHassett T. Nov.Gen. to Resolve Molecular Polyphylywithin the Aplanochytrids within the thraustochytrids. within thraustochytrids. the 3 : 18SrRNA;pollen baiting,Norwegian homoplasia; Sea, thraustochytrid cells liter per 2015).et(Ueda of al. Members Labyrinthulea are the A culture of an unknown unicellular hete haliotidis a and R. Gradinger : BHassett,UiT Norges universtiet, arktiske BFE, NFH bygget, Labyrinthulea

19077824461; E-mail: [email protected]; E-mail: a

(=Labyrinthulomycota) and the Erection of a ) , a, pathogen of abalone. Testingof phylogenetic areheterotrophs cosmopolitan thatbehave as marine and freshwater ecosystems. Members of isolate’s 18Ssmallribosomal revealedsubunit electronmicroscopy revealed complex cell Version of Record. Please as this Version cite ofRecord. article rotrophiceukaryote wasestablished A. haliotidis Penicillium brevicompactum among the the among

A. A. Accepted Article in 300mL culture flasks 4 at from asingle PmTGcell on agar medium. Axenicwere cultures maintainedin broth PmTG examined after 24hours andfungal-likeisolates peni (500 streptomycin sulfate with mg/L) and seawater (Instant , Blacksburg, VA,USA) adjustedto a salinityof33 and amended This by is protectedarticle reserved. Allrights copyright. St.Louis, MO,liter USA) 20g 5gglucose, agar (Sigma-Aldrich, tryptone, like organismswas drop-streaked onto PmTGcontaining media 1g peptonized1g milk, baited with usingtemperature/salinity a OH, Yellow Springs, probe(YSI, USA). weresamples Water Norway E18.906)on(N69.663 22May 2017.Seawater salinity was measured in lab the A seawater sample was collected from the nearshore marine environment outside of Tromsø, Isolation and culture AND METHODS MATERIALS Labyrinthulochytrium arktikum Labyrinthulochytrium useof thraustochytrid weevolution, molecular evidence and tolife history information erect Labyrinthulochytrium arktikum data, ultrastructure, and lifehistory,describe we ournew isolate aas new species, haliotidis our isolateamong the diverse an characteristics as revealedmicroscopy examination ofourisolate a lifehistory similar ultrastructural and unicellular isolate revealed closest molecular affinityto Norwegian Sea near Tromsø,Norwayusing pollen baitingtechniques. Analysis of our We investigated the presence and diversity of the free-living inLabyrinthulea the nearshore the aplanochytrids (e.g. Moro et al. 2003). (Leander &Porter2001) andbeen has subsequently excluded molecular phylogeniesoffrom molecular sequencehas suggestedbeen tobederived from thraustochytrid a contaminant involving Molecular thes ofthe18SSSU-rRNAanalysis of and ectoplasmic nets; yet, inare morphologically congruentproduction of the aplanospores. aplanochytrids (Moro et al. 2003; FioRito et al sporangium wall (Bahnweg & Sparrow 1974). There are currently nine described eucarpic thallus thatproduces aplanosporeswhich arereleased by rupturea theof The taxonomic genus systematicsLabyrinthulea &Porter (Leander 2000). (Yokoyama et al. 2007; Yokoyama & Honda 2007; FioRito et al. 2016) and guide have region(SSU-rRNA) coding been used asdiagnostic a toolto circumscribe novel clades Leander al.et 2004); consequently,sequences DNA 18S ofthe small ribosomalsubunit morphological characteristics speciesfor identification& (Damare Raghukumar 2006; net (Leander et al. 2004). Phenotypic plasticity ectoplasmicmotile the net;and aplanochytrids use that glidingmotility with ectoplasmican and examined for putative ecological roles. brevicompactum with thealga our isolate ofculturing thatbranches sistertouncharacterized isolates from Japan. Based onmolecular Aplanochytrium haliotidis Aplanochytrium Pinus Pinus was conducted at roomtemperature fo A. haliotidis sp.(Pine) pollen for 3-days atroom temperature.Pollen containing fungal- Aplanochytrium

(gen. nov.) that circumscribes o C for life history characterization and molecular analysis. Co- analysis. molecular and C characterization history forlife .analysis Phylogeneticof 18SSSU-rRNA sequences places . (sp. nov.). Tobegin robustphylogenetic forming hypotheses d homoplasious thraustochytrids

(Bongiorni et 2005;al. et FioRito al. 2016), whose socialis is morphologically-typed as having a monocentric . 2016) that differentially produce zoospores cillin G (200 mg/L) antibiotics. Plates were within these subgroups hinder the useof were harvested and repeatedly subcultured e nine species suggest Aplanochytrium haliotidisAplanochytrium

r 24hours and 72 hours, respectively A. haliotidis andthe marine ¸ formingwith aclade . Light and electron Light and . s a polyphyletic clade -1 of artificial artificial of and Penicillium Penicillium A. A. Accepted Article This by is protectedarticle reserved. Allrights copyright. werein Samples postfixed 2%OsO MgCl)and overnight 15-minutes rinsed twice sodium 0.1M with for cacodylate buffer. universal fixative (100mM cacodylateacid,4%1% formaldehyde, glutaraldehyde and2mM 4,000 x seawater threefor days. wereSamples then centrifuged in 50-mL tubes polypropylene at wereCultures in PmTG grownfor broth one week then subculturedandin 0.22 µm-filtered microscopyElectron al. 2016). recommendations.company’s wereChromatographs withexamine MEGA v7.0.26(Kumar et sequence reactionswere resolvedusing Appliedthe Biosystems 3730XL following the (Beckman Coulter, Indianapolis,IN, USA)following the company’s recommendations. The Therecommendations. sequence reactions wereusing purified CleanSEQ Agencourt’s (Thermo Fisher Scientific, Waltham, MA,USA) and cycled following the company’s amplicon. Sanger sequencing was conducted with the BigDye v3.1 Cycle KitSequencing bidirectionally Sangerto maximize confidence sequenced in base calls lengththealong ofthe purified using the PureLink Quick PCR Kit Purification (Thermo Fisher Scientific) and pairs (White al.et 1990), as previouslydescribed (Hassett et 2015).al. products PCR were AACTTAAAGGAATTGACGGAAG-3’/5’ - TCCGCAGGTTCACCTACGGA-3’) primer (5’ - NS5/NS8-3’) and CTTCCGTCAATTCCTTTAAG - 18S NS1/NS4(5’-GTAGTCATATGCTTGTCTC-3’/5’ rRNA amplified using polymerase chainreaction (PCR) Platinumwith Taq (Life Sciences) and the Hilden,(Qiagen, Germany) protocol. the following manufacturer’s GenomicDNA was DNAGenomic isolated was 14-day from cell andsequencing amplification, DNA extraction, eyepiece camera (AmScope) at 40xmagnification long with rangeobjectives. phase microscope.contrast Video wasacquired using a fixed microscopewith adapter morphologyand history life was viewed using light microscopyon aLeitz DM ILinverted Cultureswere examined andvideo-recorded for one weekfollowing broth inoculation. Cell Morphology andsubstrateutilization pseudoreplications. parsimony Maximum (MP) tree was generated thenand using bootstrapped likelihoodmaximum with(ML) 1,000 phylogeneticfor generation using tree BayesianA information criterion. neighbor joining regions weremanually removed.wasMEGA7 us databaseNCBI (Table 1) MEGA7insubsequently and alignedMuscle. using Allunalignable (MSA) sequence alignments Multiple were from DNAcreatedsequences the from retrieved analysis Phylogenetic microscopeJapan). Tokyo, (JEOL, polymerize at 60 at polymerize embedded with propyleneoxide and resin epoxy (EPON) Samples were overnight. to left 1:1 sample was rinsedpropylene with oxide times three for five minutes.Samples were each andrinsed finally in 100%ethanol twiceAfter for 10-minutes. ethanolrinsing, the dehydrated with ethanol:for 5minutes,30% followed by60%,90% and96%10 for minutes rinsed 15-minutes twice0.1Mwith for sodium g for onehour for toThe samples material. pellet were then fixedmodified using Trumps o Cone for day. Imagingwas withconducted a JEM-1010 electron

4 in 0.12M sodium cacodylate buffer for 30-minutes and

cultures using theDNeasy PowerSoilKit cacodylate buffer. Samples were serially serially were Samples buffer. cacodylate trees were estimated fromMSAs the and ed model to identifythe substitution best-fit Accepted Article This by is protectedarticle reserved. Allrights copyright. numerosus corium lamellaeprotube contentus, tubulata contineo; interdum, hyoideum informibus nucleum.circum Cellulam murus cytoplasmum ectoplasmumfilum reticulumfictus. Mitochondria cristae longus (16µm) aliquantum. (lateralis) vegetativae Zoosporumrapidus verso sub incrementum.Productio Zoosporumcum motorium flaggelum duo,(dia ovalibus4.5-12 Propagationisproductio amplifico. binarii dividit (~8) zoosporum et propagationis. μ epibioticum;sporangium vegetativaesporangium sphaeroideon plerumque subeo (dia4.5-14 Pinus pollinis consummo. Facile ius nutrie ZooBank: Labyrinthulochytrium Aplanochytrium haliotidis Basionym: Labyrinthuloideshaliotidis comb. nov. Type species the type naming scheme for thraustochytridsthe Etymology inference. sloughing with electron Distingumicroscopy. cristae, sometimes horseshoe-shaped around the nucleus. Cell wall scales observed biflagellate zoospores slightly oval (4.5-12 µm seawater,divisionis binary retarded andzoosporeproductionheightened. is Motile diameter).in nutrient In media preferentiallyby divides division; in binary shape (4.5-14 µm towhitein white mass culture; sporangium vegetative stages epibiotic; usually spheroidal in on pineSaprobic pollenonparasitic andgrown on nutrient abalone. Easily Greyish media. nucleum. circuminforme aliquantum (dia 4.5-12 zoospore productionemamplifico. Zoosporum (dia 4.5-14 alba moles; sporangium epibioticum; sporangi Pinuspollinis consummo inet haliotidae parasitica. Facileiusnutriendum esse colo. Cretacea ZooBank: Labyrinthulochytrium RESULTS comparison. AR2-19 sequences were grouped within the persubstitutions site. Ourisolate, were used inMEGA7 to assesscomparatively number the of inter and intra-genus base bootstrapped following 1,000 pseuoreplications. Maximum LikelihoodCompositemodels m). In dividit;dividitnutriendum binarii ius et inmarinis, binarii retard zoosporemavultis m). urn:lsid:zoobank.org:pub:B93152E1-7AEF-47FE-BE7C-90697BF9685A urn:lsid:zoobank.org:pub:B93152E1-7AEF-47FE-BE7C-90697BF9685A : μ labyrinth : m). Inmavultisnutriendum binarii ius m). dividit; dividitinretard marinis, binarii et Labyrinthulochytrium haliotidis = maze, inof recognition = the maze, phylum; type μ nov. gen. Gradinger, etR. Hassett B.T. arktikum Mitochondriam). cristae tubulata continet;interdum, hyoideum

L. haliotidis B.T. Hassett et R. Gradinger,sp. nov. (Broweremend 1986) Leander et al. 2004to ndum et marinum esse colo. Molesalbus; Labyrinthulochytrium ished from other genera basedon molecular , and thraustochytridisolates M4-103 and two um vegetativaeum plerumquesphaeroideonsubeo motorium motorium cum flaggelum duo, ovalibus (Brower 1986) B. T. Hassett et R. Gradinger ratus numerosus et bothrosomus subitus. in diameter). Mitochondria containing tubularcontaining Mitochondria in diameter). μ chytrium m)subfusiformum et forintra-genetic and inter =pot,recognizes

Accepted Article At theAt of time (22 field collection haliotidis = haliotidis Labyrinthulochytrium seawater,nearshore NorwayTromsø, (N69.663 E18.906). This by is protectedarticle reserved. Allrights copyright. cells began tosequentiallydissoc cluster into a more planer of cluster cells 1F). (Fig. offiveWithin minutes elongation,cluster cells remained clustered someuntil unknownphysical mechanism elongated single the round a single round cluster of approximately eight associated (Fig.1G).cells These associated underwent repeated rounds of binary division that, instead of migrating after division, formed Afterdivisionin binary nutrientmedium and subsequentsterile to transfer seawater, cells diameter. in µm 1F)20-35cells (Fig. smelled strongly offish. Cells maintainedin culture >1monthproducedfor atypicallylarge in broth culture as a of biofilm non-overlapping cells. Ingeneral,densebroth cultures by interconnected Thistheir cytoplasmic nets. ectoplasmic nets (Fig.mitotic Prolonged 1E). away Cytoplasmic threadswere parentcells. from able to interact form to reticulate independently of the sporangium. After binary daughter division, generallycells migrated in length)cytoplasmic that threads moved cytokinesis. Many cells long(~66produced µm of interphase seeminglythe of beforetermination conjoined initiating cells daughter indiameter). daughter Binaryinto divisionoccurred(4.5-14.0µm cells in rapid succession, followedby the ofaformation cleavage(Fig. furrows 1D), and subsequent binary division (Fig. 1C). Division14µm occurred after the loose consolidation of cytoplasmic material, content 1B)relative (Fig. to sinking(Fig. 1C cell capableof floating sinkingandin culture. cells Floating appeared to lesscontain intercellular reproducing byand mitosis less by frequently zoospore production.Vegetative were cells subculturing, axenic werecultures transfe Holotype Etymology clustering. and the absence of a single zoos basedmolecular on inference, the production bothrosomes.wall Cell scales observed sloughi lamellae10) electron-densethat multipleprotuberances, somecan form forming horseshoe-shapedsometimes aroundthe nucleus.wall of multilayer Cell (up comprised to cytoplasmic threads that canectoplasmic form ne settlement subsequent developmentintoand a in diameter) and appear (4.5-12 subfusiformµm in lateral view that can spin rapidly prior to retarded zoosporeand productionis heightened. biflagellate Motile slightly zoospores oval In nutrientpreferentiallydividesmedia by binary division; inseawater, binary division is diameter).in Asexualby reproductiondivision binary zoospore and (~8)release. µm (4.5-14 culture;usuallysporangiumWhite epibiotic, vegetativestages in mass spheroidal in shape on pineSaprobic pollen.Easily grown maintained and onnutrient mediaorinseawater. of growingat a temperature range between 4– 25 and wereobserved after several daysinnumerable producing zoospores. Cells werecapable attached topine pollen (Fig. 1A).Sporangia were thentransferred onto PmTGagar medium days of incubation at room temperature, spheroidal sporangia were observed epiphytically Labyrinthuloides haliotidisLabyrinthuloides : 1– Fig. 2 and GenBanksequence SSUrRNA SUB3113088

: arktikos = of the north, therecognizes location of this isolate (Brower 1986)(Brower emendLeander et 2004 al. to iate 1) (Vid. and rapidly migrate.These zoospores ranged in

May 2017)the wassalinityof seawater After27.6. three (Bower 1986)(Bower Hassett B.T. etR. Gradinger, nov. comb. rred tobrothPmTG medium rred and observedwere porangial rupture during zoospore release. of largezoospores, the absence ofcell vegetative cell. Production of long (16 µm) vegetative cell. Production(16long µm) of division produced anextensive matrix ofcells extensivematrixcould easily bemanipulated ng with electron microscopy. Distinguished ). Vegetative cells were between 4.5µm and and 4.5µm between were cells Vegetative ). ts. Mitochondria containingcristae, tubular o C. After several iterations of of iterations several After C. . Isolated from Aplanochytrium Accepted Article This by is protectedarticle reserved. Allrights copyright. (Fig. 3H)with unknown triplets fourcentral 3I),elements (Fig. were also observed. adense basal electron 3G).(Fig. bothrosome Centrioles comprisedof nine microtubule in in length) associationatoriginated with a complex lamellateprotuberances(~500nm-1µm developing daughtercells, each with a corresponding nucleus 3F). (Fig. Ectoplasmicnets micrographs captured cells dividing 3D, (Fig. E), with lamellar cell walls separating with the cell wall that weregenerally localized at ofpolar ends the cell(Fig3C). Electron walls Cell fo several,(sometimes Fig. 3B). tubularhorseshoe-shaped with mitochondria 3A), cristae and (Fig. lipid large globules that in vegetative cells enclosed a single nucleus in proximity to a single Golgi body, Transmission electronmicrographs (Fig. 3)revealed electron dense, multilayeredcell walls Labyrinthulochytrium per site when averaged across allgenera. Theaverage differencebetween the 1). (STable Comparativepairwise inter-genera analysis of was DNA 0.135substitutions base Labyrinthulochytrium base substitutions per site when averaged acrossgenera theall (excluding and 84%for MLandMP, respectively.pairwiseIntra-genera analysis of DNA was 0.0403 nodes were consistentlysupportedby higher bootstrap values greater than orequal to 94% however, clades;well-supported branching sistertoundescribed isolates(Fig.2) basaland to the aplanochytrids and tree topologies thatgroupedour isolate and likelihood,joining, maximum parsimonyphylogenies maximum producedand consistently distribution (TN93+G). The final MSA includedalignment 1,467 positions. Neighbor gammaestimatedofthe byarates 93withTamura-Nei, DNA substitution was substitution data within ourmultiple sequencealignment,MEGA indicated the most modelappropriate of thraustochytrids isolated Japanin (Ueda et2015). al. To most accurately molecularestimate haliotidis BLASTof the query isolate’s 18S gene identified (97.5%) identity closest to integrity to hyphal strands as a result of this . localized (SFigurecells 2). After three day of co-culturing, wasthere no observable loss of along hyphal strands (SFigure Cells cultured with myceliaof isolatesthe began to move away from algalcells. oneAfter day, noparasitismwas observed. theirwith cytoplasmic threads. minutes of ~30 After threads,interaction with cytoplasmic After twohours ofco-culturing with algae,our isolatesbegan tomigrate towardsthe algae socialis theTo ecology of ourisolate,explore cells were with co-cultured algae ( threads for (Vid.motility 1, 1:00 at bottom). cells. Afterthis conversion, some newly-formed vegetative cells used their long cytoplasmic motilitynormal thread-mediatedresuming beforequiescent state, was that typical mature for converting their flagella intothelong cytoplasmic threads),then temporarily entered a remainedmotile for minutes before several two possessed inand fl diameter) µm (4.5-12 size ) and a marine fungi ( (NCBI accession U21338.1), followed by94% identity toundescribed several . Within . ourphylogenetic (Fig. trees2, SFig. 3), included all genera formed ). The average difference withinthe and all other and groups all was 0.112 substitutions base 2). persite (STable Penicillium brevicompactumPenicillium 1). Sometimes, hyphal strands hyphalstrands 1). Sometimes, P. brevicompactum Thraustochytrium rmed multilamellate protuberances contiguous many rapidly spun (seeminglyand uncoiling A. haliotidis A. haliotidis agella (Fig. 1I, 12µm in length).Zoospores 12µm (Fig.agella 1I, migrated towardshyphae andlocalized isolateswere interspersed. Terminal among the thraustochytrids, theamong ) wasand explored for . Labyrinthulochytrium would swell inproximity to Chaetoceros Aplanochytrium was 0.028 Accepted Article supporting uniqueness the ofour novel genera to relative otherrecognized clades. The Botryochytrium base substitutions per site);however, ournew genus exceededthe averageof distance was slightly than greater averagethe distance ofourgenus new to all other genera (0.112 Inter-genus genetic differences among the (0.135thraustochytrids base substitutions per site) our proposed clade is comprised ofmore genetically-similar isolates, relative genera.other to the genetic differences within our new genus (0.028 base substitutions per site), suggesting among the thruastochytrids (0.0403 base substitutions per site) (STable 1) was greater than Labyrinthulochytrium characterization of these isolates will elucidate evolutionary relationships among evolutionarythe ofrelationship genus without this othergenera.circumscribing Future includeconfidentially theseisolates within the uncharacterized isolates; however, withoutlif on molecular phylogeny, possibleit thatis isolated in Japan (Ueda et 2015),al.branching Phylogenetically, ourisolates fall among new isolate, represented as describing initially the earli support data These thraustochytrid. with with 3). Theobservation ahyphal(Table of swelli release zoospore during wall cell zoosporangial a of absence the and cells vegetative-dividing haliotidis Labyrinthulochytrium and genera:thraustochytrid described the ten of it shares onlytwo out that with thraustochytrids,unique the among as it is capa thraustochytrids.new scheme, this the relative Within classification of haliotidis (i.e. historyevidence, with inconjunction This information life molecular specimen. thethat 18S sequence associated with identity molecular and to close ultrastructure, history, life similar with unicellularheterotrophic unknown isolated we an study, In this This by is protectedarticle reserved. Allrights copyright. reclassification of sequence was a2001) derived (Leander from contaminant &Porter likely inhibited earlier evolutionaryhypothesis conducted in this study; however, speculation thatthe 18SrRNA of the Labyrinthulea (Bongiorniaetal. 2005, FioRitoetal. 2016), including our in tests of problematic, consistently grouping outside the aplanochytrids in numerous molecular studies 2007).this Within paradigm, molecular evolution Labyrinthulea theamong (e.g. Damare &Raghukumar 2006, Yokoyama et al. phylogenies basedonrRNA the 18S smallsubunithave been used to guide inferences of al. 2004, Bongiorni al.et Damare2005, &Raghukumar Consequently, 2006). molecular homoplastic traits has confounded systematics that scheme Labyrinthulea.Phenotypic plasticity, convergent andevolution, taxonomy basedon because this group is definitively not part of the True Fungi, we preferentially use the naming this groupremains (e.g. uncertain FioRito Cavalier-Smith 2017, al.et 2016); however, historically been divided intomorphology-define The Labyrinthulea are(=Labyrinthulomycota) cosmopolitan heterotrophs that have DISCUSSION P. brevicompactum does not produce aplanospores)und

baseto all genera (0.110other thraustochytrid substitutions persite), A. haliotidis ), is does), Our not pigmented, (Table 2). release not protoplastand naked Labyrinthuloides haliotidisLabyrinthuloides itsand nearest-branching clades. Intra-genus genetic differences suggests thatit possess may virulence-associated properties. Labyrinthulochytrium artikum, . by the production of large zoospores of by production large size toof of approximate the zoospores A. haliotidis A. Aplanochytrium haliotidis a core group of undescribed thraustochytrids Labyrinthulochytrium e historye weinformation, are tounable er suppositions er suppositions made by Bower (1986) when erscores classification the of Labyrinthulochytrium sister toisolates AR2-19and M4-103.Based ngs prolongedand association ourisolate of ble ofble reproducingtrait by (a binary division d taxa. Thephylum level classification of (= are reflective of naturalare reflective of taxa(Leanderet was likely derived from the original type the likely from was derived Aplanochytrium haliotidis Aplanochytrium A. haliotidis A. is readily is from differentiated circumscribes these these circumscribes ; consequently, we; consequently, surmise has been phylogenetically or concisely describe describe concisely or L. haliotidis haliotidis L. Aurantiochytrium A. haliotidis )a as close is still is asa A. A.

Accepted Article Bahnweg,G.K. F. &Sparrow, 1974.Four new of species LITERATURE CITED microscopy. acknowledge AugustaAspar andRandi Olsenat UiT fortheirassistance with electron forhisCavilier-Smith interpreting helpThomas electron micrographs. We towould like the Seasonal Ice Zone Ecology (SIZE) working group. We would like to acknowledge wouldWeto like acknowledgeNorwegian the Research Council for funding through support ACKNOWLEDGMENTS carbon cyclingand the evolutionaryrelationship among its clades. Future research is needed to understand the ofcontributions the Labyrinthulea to marine a when a parasite as behave could and year the Bongiorni, L., Jain, L., R.,Bongiorni, S., Raghukumar,&Aggarwal, R. K.2005. Bennett, R. M., Honda, D., Beakes, G. W., & Thines, M. 2017. Labryinthulomycota. to maintain diverse and well-studied thraustochytrids.Therange of temperatures inwhich we were able importance as a parasite of abalone, while characterizing a novel branch of life among the This research helps redefine the taxonomically enigmatic appearances across various taxa. occurredintermediate or form, in the distal (i.e.9 configuration +4);however, itis possiblealso that this wascentriole either an possible thatthese central elements are microtubulesthat represent a potentially novel observed with centrioles microtubulenine triplets withfour unknown elements.It central is (Bower 1986);Golgibodies, nuclei, vacuoles and (Moss 1985).In several cross sections, we bothrosomes (Moroet al. 2003;Iwata al et for the thruastochytrids (Darley al.et 1973;1982);Kumarnumerous electron-dense Labyrinthulea.the observed We cell walls madelamella, which ofelectron-dense are typical Electron microscopy of Labyrinthulea. conjunctionwith molecular data,understand to relationshipsevolutionary among the used extensively toguide systematics (e.g.et Wyngaert al. 2017)and may proveuseful, in respectiveclades. Amongbasal-branchingthe True zoospore Fungi, ultrastructurehas been resolve enigmatic species complexes and identify true amorphic attributes thatcorrespond to This by is protectedarticle reserved. Allrights copyright. the within relationships that researchfuture employ concaten multigene resolve deep-branching relationships and to form robust inferences of evolution, we suggest backbone ofour phylogenetictreeswas considerablyless supported thannodes. terminal To . 2nd ed. 2ndPublishing,International Springer Protists. Gewerbestrasse. 14:507-542. J.M.,Archibald, Simpson, A.G. Slamovits,C. B.,H.(ed.), The Handbook ofthe ecosystems. withregions, notesof the ondistribution zoosporic fungi in the Antarctic marine Goa, India.Goa, gaertnerium L. arktikum sp.nov.:new a stramenopilan thraustochytrid protist mangrovesfrom of Am.Bot. J. suggeststhat this may organism be ecologically relevantthroughout , 156:303-315., L. arktikum Labyrinthulea , 61:754-766. , 61:754-766. revealedtypical ultrastructural features associated with

(e.g. Cavalier-Smith etCavalier-Smith(e.g. al. 2014, Kang etal.2016) to . 2016);horseshoe-shaped,. tubular mitochondria lumen - a highly variable region with numerous numerous with region variable ahighly - lumen ssociated with temperature-stressed symbionts. ated phylogeniesated to test the evolutionary L. haliotidis Thraustochytrium Thraustochytrium that is ofecological from Antarctic In: Accepted Article Doi, K., Honda, D. 2017. Proposal of Cavalier-Smith, T. 2017. Chromista and its eight phyla: a new synthesis Iwata, I.,Kimura,Motomura, Koike, K., Iwata, Honda,Y.,K., T., Tomura, K., Kanae, D. 2016. FioRito, Leander,R., C., B.2016.Leander, Characterization ofthree novel species of Damare,V., Raghukumar, S. 2006. Morphology physiology and marine ofthe straminipilan Kumar, S.Kumar, R. 1982.of Fine thestructure thraustochytrid Kang, S.,Tice, A.K. Spiegel, F. W.,Silberman, J.D., Pánek, T., Leander, C.A., Porter, D. 2000. Redefining genus the Aplanochytrium(phylum Jones, E Alderman, G.,D..B. 1971. J. This by is protectedarticle reserved. Allrights copyright. Brower, S. M. 1986. Kumar, S., K. 2016. G., Stecher, Kumar, Tamura, Darley, M.,W.Porter, D., M. Fuller, S. 1973.Cell wall composition andsynthesisGolgi- via T.,Chao,Cavalier-Smith, E.E. E., Snell, A., Berney,A.Fiore-Donno, C., M., Lewis,R. Hassett, B. T., L B.T., Hassett, (, ) Labyrinthulomycetes) for former (Stramenopiles, emphasizing protein targeting, periplastid cytoskeletaland periplastid evolution, and deep ofdeep Amoebae.evolution A.,Lahr, Kudryavstev, D. J.G, Brown, M.W. 2016.Between poda and a hard test: the Kosakyan, A., Alcântara, D.M., Roger, A.J., Shadwick,L., L. Smirnov.A,, settlement. zoospore during Stramenopiles) Bothrosome formationin 163:170. isolatedLabyrinthulomycota fromsea ochre stars ( morphological features and phylogenetic relationships. onnote theaplanochytrids fungi, isolatedthe from Indian equatorial Ocean. 81:71-85. Labyrinthulomycota). Labyrinthulomycota). biflagellate fungus. Protist isolatedthe the in Beringsub-Arctic Sea. Mesomycetozoea from sphaeroformids Can. J. Zool. J.Can. facility. mariculture ColumbiaBritish in a abalone small juvenile ofparasite pathogenic thallus and formationthe of amoeboidstage. directed scale formation in the marine eukaryote, 35:326-340. (animals, c fungi, Multigene 2014. eukaryote phylogeny reveals the likely protozoanof ancestors ancient divergences. Analysis version 7.0. for bigger datasets. datasets. bigger for 7.0. version Analysis , 166:310-322. Thraustochytrium Ó pez, J.pez, Gradinger, A., R. 2015. species Twonew of marine saprotrophic , 65:1996-2007. , Labyrinthuloides haliotidis Nova Hedwigia Protoplasma Mycotaxon Schizochyrium aggregatum sp. Mol. Biol. Evol. Arch. Mikrobiol., hoanozoans) and Amoebozoa.hoanozoans) and Monorhizochytrium globosum , 76:439-444. , Althornia crouchii , DOI:, 10.1007/s00709-017-1147-3. , 21:381-400. MEGA7: Molecular Evolutionary Genetics Mol. Biol. Evol. Protist n. sp. (Protozoa: Labyrinthuomorpha), a Can. J.Bot. , 34:2258-2270., 90:89-106. Schizochytrium aggregatum Thraustochytrium globosum Pisaster ochraceus , 168:206-219. Ulkenia amoeboidea gen. etsp. nov.,a marine (Labyrinthulomycetes, Phycol. Res. , 60:1092-1102. , , 33:1870-1874. , Č Mol. Phylogenet. Evol. epi gen. nov., com. nov. nov. com. nov., gen. č ka, I., Kostka, M., , 65:188-201. Indian J. Mar. Sci. Mar. Indian J. ). ). . I. Vegetative Vegetative I. . Mar. Biol.Mar. , with a based on , , , Accepted Article Moro, I., Negrisolo,Moro, I., E., Callegara,A.,2003. Andreoli, C. This by is protectedarticle reserved. Allrights copyright. Yokoyama, R. & Honda D. 2007. rearrangement Taxonomic ofgenusthe Wyngaert,S. V. D., K., Seto,Rojas-Jimenez, K.,M. Kagami, 2017. A new parasitic chytrid, White, T., Lee, B., Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal M.,Ueda, Nomura, Y., Doi,Nakajima, K., M., Honda,D. 2015. Seasonal dynamics of C.K.Tsui, Marshall, M., W., Yokoyama,R., Honda, J. D., Lippmeier, C., Craven, K. D., F.Sparrow, K. 1960. Aquatic Phycomycetes. University of Michigan Press,Ann Arbor. Moss, S. T. Anultrastructural 1985. studyof taxonomicallysignificant characters ofthe Leander, C. Porter,A., D., Leander, B.S. 2004. Comparative morphologyand molecular Leander, C. Porter,A., D. 2001. Labyrinthulomycota of The is comprised threedistinct Yokoyama,Salleh, R., B., Honda, D. 2007. Taxonomicthe rearrangement ofgenus

freshwater desmid Staurastromyces oculus 38:315–322. AGuide PCR Protocols: toMethods andApplications. Academic Press,Orlando. RNA Genes Phylogenetics. for waters. coastal thraustochytridsculturable (Labyrinthulomycetes, Stramenopiles) in estuarine and Phylogenet.Mol. Evol. the forimplications evolutionary loss ofchloroplasts ofand gain ectoplasmic gliding. P.Peterson, D., Berbee, L.M. 2009.Labyrinthulomycetes phylogeny and its Thraustochytriales and the Labyrinthulales. 340. fromLabyrinthulomycota the Southern Ocean (Ross Sea, Antarctica). phylogeny aplanochytrids of (Labyrinthulomycota). lineages. and erectionand of phylogeny (Thrasutochytriaceae,Labyrinthulomycetes): emendation for sensu latobasedon morphology, chemotaxono Mycoscience 211. erection oferection phylogeny (Thraustochytriaceae,Labyrinthulomycetes): emendation for senu lato based on morphology, chemotaxonomical characteristics, and 18S rRNA gene Mycologia Botryochytrium , 48: 329-341. , Aurantiochytrium Aquatic Microb. Ecol. Staurastrum , 93:459-464. , , 50:129-140. , (Rhizophydiales, Staurastromycetaceae nov.),infectingfam. the , Parietichytrium sp. In : Innis, M., Gelfand, D., Sninsky,J., White, T.(ed.) and Protist , 74:187-204. Oblongichytrium , 168:392-407. , and Bot. J. Linnean Soc. mic characteristics, and18S gene rRNA Sicyoidochytrium Sicyoidochytrium Eur. J. Protistol. Eur. J. Aplanochytrium stocchinoi gen. nov. nov. gen. , 91:329-357. Mycoscience gen. nov. nov. gen. Schizochytrium , 40:317-328. , Protist Schizochytrium Ulkenia , 154:331- , , 48:199- , Ulkenia : a new : a and

Accepted Article along the hyphae of LightFigure S1. microscopy showing at400xmagnification the association of INFORMATION SUPPORTING Table 3.of morphological Summary between differences Table 2.Diagnostic table thatdetailsmorphological differencesbetween genera. described hypotheses. Table 1.GenBank accession numbers of18SrRNA loci used to testevolutionary withmicrotubule unknown triplets four elements.central proximity to a lamellarprotuberance.Thecentriole the on left is comprised ofnine protuberances, one protuberance is in close pr association with the(white bothrosome a inclusion localizeda to vacuole, a and mitochondria. G) Complex lamella in protuberance cell wall, each cell with itsown nucleus. Also observable is a Golgi body, electronan dense thetovacuoles. completely-cleaved (EDI) localized F) Three cells enclosed incontiguous a section separatedby electron-dense,an newly- formed, electron dense, thin cellwall (CW). E) Diving cellsplitting into threesection, each strands.Cell D) undergoing with binary division nuclei, two mitochondria, and a newly- cellVegetative awith lamellar(L protuberance Small vegetative cell filled with lipidfour globules in association with a mitochondria. C) proximity to the nucleus, andhorseshoe-shaped vegetativeof a cell with a nucleus (N)containingnucleolus a (NU), a Golgi body (G)close in This by is protectedarticle reserved. Allrights copyright. valueslikelihood pseudoreplications. following 1,000 genetically similar isolates to ExpandedFigure S3. phylogenetic showtree informing proximity to localized LightFiguremicroscopy S2. at1,000x magnification of Fig. 3.of electron Transmission micrographs Labyrinthulochytrium haliotidis pseudoreplications. Shadedillustrates area our new species thatbranches sister to nodes displaying maximumparsimony/maximum likelihood values following 1,000 Fig. 2. Condensed, rooted phylogenetic tree I)dispersal. Two zoospores, onedisplayingbiflagellation. zoospore development. H) Cellclusterpriorand following elongation to sequential zoospore dividing.cell F)Largeweeks several after in G) culture. of Cluster formed cells during withPmTG media.cells in cyt E)Cells ofseveral generations division binaryillustrating spacing variation and ofsize. D)Dividing cytoplasmic inclusions than sinking cell varieties. C) Representative vegetative cells after visible less with water of the surface the to coverslip aglass by touching recovered cells A) Pine pollen (p) with10µm. Fig. 1. Light microscopy of LEGENDSFIGURE P. brevicompatcum Labyrinthulochytrium arktikum L. arktikum L. arktikum . L. arktikum .

oplasmic threads forming ectoplasmic net,while rrow). H)Vegetative cellrrow). lamellar withtwo

displaying maximum parsimony/maximum parsimony/maximum displaying maximum attached(white epibioticallyB) arrow). Floating ing representative genera and the most of unknown anddescribed thraustochytrids with

oximity to a centriole. I) Two centrioles inTwo a to oximity centriole. I) centrioles cells L. arktikum formed cellformed wall, denseelectron inclusions P) comprised of contiguous electron denseof contiguous electron comprised P) mitochondria with tubularcristae (M). B) . P. brevicompactum L. haliotidis L. haliotidis . A)representative A cross section . Black scalebar represents and L. arktikumL. hyphal swellings L. arktikum . Accepted Article This by is protectedarticle reserved. Allrights copyright. inmotility seawater. Movie Light S1. microscopy capturingsequential video zoospore dispersal and sporangium Likelihood model. between groups are Analysesshown. were Table S2. Thenumberbasesubstitutions of persitefrom averaging sequence overall pairs modelLikelihood inMEGA7. within each groupshown. are Analyseswereconducted using the Maximum Composite Table S1. Thenumberbasesubstitutions of persitefrom averaging sequence overall pairs conductedusing the Maximum Composite Accepted Article This by is protectedarticle reserved. Allrights copyright. GenBank Accession Numbers Ingroup: Isolate/Taxon Name hypotheses. Table 1.GenBank accession numbers of18SrRNA loci used to testevolutionary Aurantiochytrium Aurantiochytrium Aurantiochytriumlimacinum Aurantiochytrium Aplanochytrium Aplanochytrium Aplanochytrium Aplanochytrium Aplanochytrium Aplanochytrium Aplanochytrium Aplanochytrium Aplanochytrium Aplanochytrium Aplanochytrium Aplanochytrium Aplanochytrium Aplanochytrium haliotidis Aplanochytrium Aplanochytriumstocchinoi Aplanochytriumminuta kerguelense Aplanochytrium Aplanochytriumblankum Labyrinthula Labyrinthula Botryochytriumsp. Botryochytriumradiata Botryochytriumprofunda Botryochytriumradiatum Parietichytrium Parietichytrium Parietichytrium sarkarianum Parietichytrium Parietichytrium sarkarianum Labyrinthula zosterae Labyrinthula Labyrinthula Thraustochytriidae sp. SEK 690 AB973545.1 AB973545.1 sp. Thraustochytriidae SEK AB973546.1 690 sp. Thraustochytriidae SEK 691 Thraustochytrium Thraustrochytrium Thraustochytriidae sp. M4-103 AB073307.1 AB073307.1 Thraustochytrium aureum sp. Thraustochytriidae M4-103 AB973547.1 sp. Thraustochytriidae SEK 692 Labyrinthulochytrium arktikum arktikum Labyrinthulochytrium p 7 AB220158.1 sp. L59 sp. L72 sp. N8 sp. N12 s.S15 EU851170.1 EU851173.1 EU851169.1 sp. S2125 EU851167.1 sp. S2128 EU851168.1 sp. S2124 AF348520.1 FJ810216.1 sp. S1961 sp. S19610 KT901795.1 sp. SC1-1 FJ799799.1 sp. S1a AF348516.1 sp. APKK4 AF348518.1 sp. PBS07 AF348517.1 sp. PR1-1 AF348521.1 sp. PR15-1 EU851171.1 sp. PR12-3 sp. SC24-1 sp. SC2129 sp. H1-14 sp. F3-1 sp. MBIC11085 sp. sp. 18W AB973565.1 sp. RCC893 p R-9 AB810950.1 sp. AR2-19 p R-9 AB810952.1 sp. AR2-19 143 DQ023614.1

18W-15a

U21338.1 AJ519935.1 L27634.1 AB022103.1 KX160007.1 MG099001 AB811030.1 AB973564.1 AB022115.1 AB022114.1 AB355410.1 AB811029.1 AB073304.2 AB355411.1 AB290580.1 AB244715.1 FR875311.2 AB246794.1 AB246795.1 AB095092.1 AB022110.1 AB073305.2

Accepted Article This by is protectedarticle reserved. Allrights copyright. Ulkenia Ulkenia Sicyoidochytrium minutum Sicyoidochytrium Sicyoidochytrium minutum Schizochytrium Schizochytrium Schizochytrium Schizochytrium aggregatum Schizochytrium Thraustochytrium striatum Thraustochytrium striatum Thraustochytrium pachydermum Thraustochytrium aggregatum Thraustochytrium gaertnerium Thraustochytrium kinnei Euplotidium arenarium triquetra Heterocapsa Chromeravelia Outgroup: Ulkenia profunda Ulkenia visurgensis Ulkenia visurgensis sp. ATCC28207 sp. SEK 214 s.SK35 AB290577.1 AB290578.1 AB290576.1 sp. SEK 345 sp. SEK 346 sp. SEK 210 sp. AB052556.1 sp. 141

AB290355.1 AB290585.1 AB183659.1 AB355412.1 AB022106.1 AB022112.1 KT598545.1 AB022113.1 AB022109.1 AY705753.2 L34668.1 L34054.1 DQ100296.1 AB022116.1 AB022104.1 Y19166.1 GU594638.1 JN986791.1 Thraustochytrium Sicyoidochytrium Schizochytrium Parietichytrium Monorhizochytrium Labyrinthulochytrium Japonochytrium Botryochytrium Aurantiochytrium Althornia Ulkenia Accepted Article Reference release Protoplast division Binary thallus Pigmented This by is protectedarticle reserved. Allrights copyright. 1 Table 2.Diagnostic table thatdetailsmorphological differencesbetween genera. described Thraustochytrium aggregatium o Yokayama et al. 2007 Yokayama et al. 2007 Yokayama et al. 2007 Doi& Honda 2017 Yokayama et al. 2007 Yokayama & Honda 2007 No Jones1971 &Alderman No No No Yes No No No Yes No No Sparrow Yes 1960 o e Yokayama et al. 2007 Yes No is onlythe exception 1 - Doi & Honda 2017 Accepted Article arktikumL. haliotidis L. Forms clusters 3.of morphological SummaryTable No Yes 4 3 Z d d .5-12 µm Yes No No Yes Yes Yes µm .5-12 µm .7-7.1 ifferences between between ifferences oospore diamete oospore r Binary division L. haliotidis and L. arktikum L. rupture Zoospora . n n gial

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