Thraustochytrium gaertnerium sp. nov.: a New Thraustochytrid - CCMB

ARTICLE IN PRESSProtist, Vol. 156, 303—315, August 2005http://www.elsevier.de/protisPublished online date 3 August 2005ORIGINAL PAPERThraustochytrium gaertnerium sp. nov.: a NewThraustochytrid Stramenopilan Protist fromMangroves of Goa, IndiaLucia Bongiorni a,1 , Ruchi Jain a , Seshagiri Raghukumar a,2,3 , and Ramesh Kumar Aggarwal ba National Institute of Oceanography, Dona Paula, Goa — 403 004, Indiab Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad — 500 007, IndiaSubmitted December 22, 2004; Accepted May 19, 2005Monitoring Editor: Míchael MelkonianThraustochytrids are ubiquitous, chemo-organotrophic, marine stramenipilan protists belonging tothe class Labyrinthulomycetes. Their taxonomy is largely based on life cycle development stages. Wedescribe here a new species of thraustochytrid isolated from mangroves of Goa, India. The organismis characterized by large zoosporangia with two distinct development cycles. In one, typical thalli withectoplasmic net elements mature into zoosporangia that divide to form heterokont biflagellatezoospores, leaving behind a proliferation body. In the second type, the thalli develop into amoeboidcells, reminiscent of the genus Ulkenia Gaertner. Unlike Ulkenia, however, the ‘amoebae’ do notimmediately produce zoospores, but round up prior to division into zoospores. The two types ofdevelopment occur simultaneously in single cell-derived in- vitro cultures. Molecular characterizationof the new isolate involving 18S rRNA gene typing and comparative phylogenetic analysis furtherestablish it to be a new and distinct thraustochytrid species with Schizochytrium aggregatumGoldstein and Belsky and Thraustochytrium kinnei Gaertner as the closest forms. We have named thisnew species as Thraustochytrium gaertnerium, deriving its species name in honour of Dr AlwinGaertner, a pioneer in the studies of taxonomy and ecology of thraustochytrids.& 2005 Elsevier GmbH. All rights reserved.Key Words: Thraustochytrium gaertnerium sp. nov.; thraustochytrids; Straminipila; mangroves; India.IntroductionThraustochytrids are a widespread group ofmarine fungi, belonging to the Kingdom Chromista(Cavalier-Smith 1993; Cavalier-Smith et al. 1994)1 Department of Marine Science, Polytechnic University ofMarche, Via Brecce Bianache, 60131, Ancona, Italy2 Corresponding author;e-mail raghu@darya.nio.org (S. Raghukumar)3 Present address: 313, Vainguinnim Valley, Dona Paula, Goa403 004, Indiaor Straminipila (Dick 2001; Raghukumar 2002).Typically chemoorganotrophic and morphologicallyresembling chytridiaceous and oomycetanfungi, they were originally placed among thephycomycetous fungi (Sparrow 1960) or the KingdomFungi (Eumycota of Ainsworth et al. 1973),prior to their transfer to the Straminipila. Thraustochytridsdiffer from members of the KingdomFungi in several ways. Their cell walls are multilamellate,made up of dictyosome-derived circular& 2005 Elsevier GmbH. All rights reserved.doi:10.1016/j.protis.2005.05.001

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ARTICLE IN PRESSA new Thraustochytrid from Mangroves 307behind. Mature thalli in the second type becomingamoeboid. Amoebae 25—40 mm in length and12.5—27.5 mm wide, with a hyaline, sheath-likeectoplasm and a granular endoplasm, exhibitingvery slow movements. Amoebae rounding up toform zoosporangia as above. No proliferationbody remains. Zoospores measuring 5.5—7.3 mmin length and 3—4.5 mm wide.Life cycle and TaxonomyYoung vegetative cells resulting from freshlyencysted zoospores measured around 4—5 mmin diameter (Fig. 1). In pine pollen cultures, suchcells developed into mature thalli in about24—25 h. During the course of development (after18 h), young thalli of 10—15 mm diameter werefilled with prominent droplets (Fig. 2). Two differenttypes of development were observed. In Type I,the young thallus developed directly into a maturethallus. At the onset of maturity, prior to zoosporeformation, the contents of the mature thallus, thezoosporangium, became granular in appearance(Fig. 3). The zoosporangium was characterized bya thin cell wall and measured 15—30 mm indiameter. The first sign of zoospore formationwas the appearance of an undulate outer marginof the zoosporangial contents (Fig. 4). Cleavage ofzoospores then took place rapidly and outlines ofthe zoospores became visible (Figs 5, 6). Theproliferation body was clearly visible at this stage(Fig. 7). Prior to their liberation, the zoosporesshowed rapid movements inside the zoosporangiumfor a very short period. Zoospores wereinitially released through a tear in the cell wall (Fig.8). As zoospore release progressed, most of thecell wall disintegrated totally (Figs 9, 10). A largeproliferation body, 5—10 mm in diameter was left,following the release of the zoospores (Fig. 10).The zoosporangia produced 10—75 zoospores.Zoospores were bean-shaped and possessed twoflagella (Fig. 11). The flagella were apicallyattached, the long anterior and the shorter posteriorlocated one below the other. The zoosporesmeasured 3—4.5 5.5—7 mm in size, with alength-to-width ratio of 1.4:1 to 2:1. The duration,starting from the time of zoospore initiation till therelease of all the zoospores was approximately60 min. Amoebae and zoospores were alsoobserved in the agar medium. Prominent,branched ectoplasmic net elements were producedboth in pine pollen culture and MV agarmedium (Fig. 12). Colonies of the thraustochytridin MV agar were circular to subcircular andcontained large, globose, hyaline cells (Fig. 13).In addition to the above mode of zoosporeformation, an amoeboid mode (Type II) was seensimultaneously in cultures. Some of the maturethalli, instead of developing into zoosporangia,began to assume an amoeboid shape (Figs 13,14). This transformation into amoebae was rapidand was complete in less than 1 minute. Such‘amoebae’ ranged from 25 to 40 mm in length and12.5—27.5 mm in width. They were characterizedby a very prominent, hyaline and sheath-likeectoplasm, while the rest of the cell was denselygranular (Fig. 15—17). The amoebae were capableof rapid transformation of shape and moved veryslowly (Figs 15—18). The amoeboid stage lastedabout 15 min. We did not detect any bacterivoryby such amoebae in cultures contaminated withbacteria. Subsequently, the amoebae rapidlyrounded up into cells that had the same dimensionsas normal zoosporangia (Figs 18—20).Rounded up protoplasts were smaller than theamoebae, presumably because the latter wereflat. At this stage the presence of the cell wall thathad earlier surrounded the cell could be discerned(Figs 21—24). These cells, the zoosporangia of‘Type II’ divided into zoospores in a manner similarto those of ‘Type I’ (Figs 21—25). No proliferationbodies were detected in the Type II zoosporangiaat the end of zoospore division (Figs 26, 27). Thenumber of zoospores produced by these wassimilar to those of Type I and no differences werenoticed in the zoospore shape. Numerous amoeboidcells were seen at the bottom of the Petriplate in seawater/pine pollen cultures of about4—7 days. During the early stages of establishingaxenic cultures, numerous cigar-shaped, limaxamoebae were regularly observed in the culturesThese were not subsequently noticed in seawater/Figures 14—27. Thraustochytrium gaertnerium sp. nov. Type II Developmental stages. 14. Maturezoosporangium. 15. Transformation of mature cell into an amoeboid cell. 16—18. Rapid changes in shapeof the amoeboid cell. Arrow in Figs 17 and 18 indicate the ectoplasm. 19—20. Rounding up of the amoeboidcell. 21. Initiation of zoospore cleavage of the rounded up amoeboid cell. 22—24. Mature zoosporangium.Note the presence of a loose cell wall. 25—27. Zoospore release. Figs 16—27 are from the same cell. Arrowsin Figs 22—25 indicate the remnants of a cell wall. Bar in Figure 14 represents 20 mm and is common to Figs14—27.

ARTICLE IN PRESS308 L. Bongiorni et al.pine pollen cultures. However, in one instance,when a culture grown on MV broth was transferredto the continuous flow chamber, as mentionedunder ‘Methods’, numerous normal, globose cellstransformed into such limaciform amoebae within3 h. Such amoebae measured 22—26 mm in lengthand 3.6—5.5 mm in width and also rounded uplater to form Type II zoosporangia. Motile zoosporeswere observed in cultures up to 1 monthold. Limaciform amoebae are not unusual amongthraustochytrids. Honda et al. (1998) have describedvery similar amoebae in the life cycle ofSchizochytrium limacinum. Such amoebae alsoform part of the life cycle of Ulkenia amoeboidea(Raghukumar 1980).The behaviour of the thraustochytrid in a rich,organic medium, the MV broth, did not differ fromthat in seawater/pine pollen cultures. Type Izoosporangia, amoebae and Type II zoosporangiawere produced also in MV broth. However, thezoosporangia were much larger and measured17.5—50 mm in diameter. Zoospore numbersreached values of 20—80 per sporangium.Phylogenetic analyses based on the 18S rRNAgene sequence variation, irrespective of theclustering algorithm (NJ and ML), robustly establishthe protist species analysed and described inthis study, to be a new member of the Labyrinthulomycetes,genetically closest but significantlydistinct from all other referencethraustochytrids compared in the analysis (Figs28, 29; Table 2). The overall topologies of differentphenetic trees obtained in the study were similarto each other except for minor differences in thebranch lengths. Further, the phylogenetic analysisrevealed three broad lineages of the comparedtaxa, as has been observed earlier by others,namely the labyrinthulids, aplanochytrids and thethraustochytrids (Honda et al. 1998; Leander andPorter 2001). The phylogenetic tree shows thatwithin the lineage comprising the thraustochytrids,species of the 3 major genera, Thraustochytrium,Ulkenia and Schizochytrium are interspersedamong each other. A similar observation has beenmade by Honda et al. (1998). This suggests thatthe major morphological characters on which thethraustochytrid taxonomy is based are homoplasious.In other words, binary division, the amoeboidstage, or division within zoosporangia mighthave evolved independently several times withinthe thraustochytrids. This makes the classificationof thraustochytrid species difficult, when basedsolely on morphological characters. In the presentcase, the species morphologically resembles thegenus Thraustochytrium Sparrow in its Type Idevelopment and Ulkenia Gaertner in its Type IIdevelopment. In Thraustochytrium, the contents ofthe zoosporangium divide into zoospores whilestill enclosed within the rigid cell wall. Zoosporesare liberated by a tear in the wall or its partial ortotal disintegration. Gaertner (1977) erected thegenus Ulkenia to include species in which thecytoplasmic contents are released as a nakedprotoplast either because of a total disappearanceof the cell wall or through the escape of theprotoplast through an opening in the cell wall. Theprotoplast then undergoes amoeboid movements,prior to division into zoospores. The amoeboidmovement of the protoplast in the Type II developmentof the present species resembles that inUlkenia. However, the protoplast subsequentlyrounded up prior to division into zoospores anddid not undergo divisions while the amoeboidstage persisted, as in the genus Ulkenia (Gaertner1977). Besides, the cell wall of the zoosporangiumappeared to have persisted throughout, becomingevident when the ‘amoebae’ rounded up (Figs21—24). Molecular phylogenetic analyses provideda clearer picture of the taxonomic positionof this species. In all the phenetic trees Thraustochytriumgaertnerium appears as a distinct newmember of the thraustochytrid group with T. kinneias the closest known species of the GenusThraustochytrium. However, the sister taxon (andthus the closest relative) to T. gaertnerium is agroup of four taxa comprising S. aggregatum and3 other thraustochytrids whose accession nos. aregiven in Table 2. The present species is beingdescribed as a new species of Thraustochytium,based both on the 18S rRNA gene sequences andthe Type I development.The genus Thraustochytrium Sparrow is characterizedby monocentric, eucarpic, epi- andendobiontic thalli that produce ectoplasmic netelements. The entire contents or part of thecontents of the mature thalli, the zoosporangiadivide into zoospores. Zoospores are characteristicallyheterokont, with a longer apical, tinsel andshorter, posterior whiplash flagellum. One or moreproliferation bodies may be produced. Zoosporesare liberated by a tear in the wall or a partial ortotal disintegration of the cell wall of the zoosporangia.The proliferation bodies persist after zoosporesare liberated. Morphologically, the presentspecies resembles Thraustochytrium aureumGoldstein (Goldstein 1963a), T. motivum Goldstein(Goldstein 1963b), T. kinnei Gaertner (Gaertner1967) and T. benthicola Raghukumar (Raghukumar1988) in the production of a single proliferationbody. The proliferation body in T. motivum and T.

ARTICLE IN PRESSA new Thraustochytrid from Mangroves 309Figure 28. Phylogenetic analysis of the thraustochytrids, aplanochytrids and labytrinthulids bases on themultiple alignments of 18S rDNA sequences. The Neighbour joining tree is constructed using Kimura 2-parameter. The numbers at each internal branching shows bootstrap values only for the nodes supported bymore than 50%. (500 replicates). The numbers on the branches are the branch lengths. Bar represents 0.065mutations per site.kinnei is distinctly large and formed much beforethe zoospores are cleaved. The sporangia arepyriform to ovate as compared to the globose tosubglobose zoosporangia of T. gaertnerium andfewer zoospores are produced. Thraustochytriumbenthicola produces irregularly shaped zoospor-

ARTICLE IN PRESS310 L. Bongiorni et al.Figure 29. Phylogenetic analysis of the thraustochytrids, aplanochytrids and labytrinthulids bases on themultiple alignments of 18S rDNA sequences. The phylogram is the best maximum likelihood tree ðlnðLÞ ¼10234:017Þ and distance is calculated using gamma corrected Kimura 2-parameter. The numbers at eachinternal branching shows bootstrap values only for the nodes supported by more than 50%. (500 replicates).The numbers on the branches are the branch lengths. Bar represents 0.089 mutations per site.

ARTICLE IN PRESSA new Thraustochytrid from Mangroves 311angia. T. aureum is distinct from the presentspecies in that it produces smaller zoosporangia(8—17 mm) and contains fewer zoospores (lessthan 50). None of the above species has thespecial Type II development found in T. gaertnerium.Schizochytrium aggregatum which appears ina sister clade to T. gaertnerium is morphologicallya distinct genus, characterized by repeated binarydivisions of the vegetative cells. The following keydistinguishes T. gaertnerium from other species ofThraustochytrium that produce one or moreproliferation bodies.seawater were poured in sterile 5 cm Petri dishes.A small amount of pine pollen, sterilized in an ovenat 100 1C for 48 h was added as ‘bait’. The plateswere incubated at room temperature (approximately28—30 1C) and were examined for growthof thraustochytrids after 3—7 days. The organismwas isolated in axenic cultures by subculturing inseawater/pine pollen as above, but with anaddition of 0.075% streptomycin and 0.036%penicillin. Absence of bacteria in this culture wasconfirmed microscopically and also by streakingthe culture on Modified Vishniac’s (MV) agar plates1. Single proliferation body produced.................................................................................................... 21. More than one proliferation body produced...................................................................................... 82. Proliferation body delineated prior to zoospore formation; prominent............................................. 32. Proliferation body delineated during zoospore formation; not prominent......................................... 63. Zoospores nonmotile at the time of discharge, lacking flagella; developing flagella subsequently.................................................................................................................... Thraustochytrium proliferum3. Zoospores motile even at the time of discharge, with fully formed flagella...................................... 44. Cell wall mostly persistent, or persistent as a small collar following zoospore discharge.................54. Cell wall totally disintegrating following zoospore discharge............................................................................................................................................................................ Thraustochytrium antarcticum5. Zoosporangia globose, subglobose or obpyriform, most of the cell wall persistent after zoosporeliberation.................................................................................................... Thraustochytrium motivum5. Zoosporangia obpyriform, most of the cell wall disintegrates during zoospore liberation, leaving justa collar around the zoosporangium............................................................... Thraustochytrium kinnei6. Zoosporangia smaller than 20 mm ; not more than 50 zoospores per zoosporangium; no amoeboidstages produced during life cycle..................................................................................................... 76. Zoosporangia often up to 30 mm diam; up to 75 zoospores produced; Amoeboid stages presentduring life cycle................................................................. Thraustochytrium gaertnerium sp. nov.7. Zoosporangia irregular in shape; zoospores ovoid; flagella apical and subapical............................................................................................................................................. Thraustochytrium benthicola7. Zoosporangia up to 17 mm in diam; less than 50 zoospores produced; flagella lateral.......................................................................................................................................... Thraustochytrium aureum8. Number of proliferation bodies not more than 4, zoospores motile at discharge..................................................................................................................................... Thraustochytrium multirudimentale8. Number of proliferation bodies more than 4....................................................................................109. Number of proliferation bodies 5—50; zoospores not motile at the time of discharge.............................................................................................................................................. Thraustochytrium rossii9. Number of proliferation bodies 3—10; zoospores motile at discharge........................................................................................................................................................... Thraustochytrium kerguelensisMethodsThe protist analysed and described in this studywas isolated during April 1999 from waterscollected from Chorao mangroves in Goa (Lat:15127’N; Long: 73148’E). Samples were collectedusing sterile glass vials and brought to thelaboratory within 1 h for isolation using the pinepollen baiting method (Gaertner 1968; Porter1990). About 3 ml water sample and 2 ml of sterile(Perkins 1972). Bacteria-free colonies were subculturedin seawater/pine pollen cultures. A singlepine pollen grain with a thraustochytrid cellattached to it was picked up using a fine glassloop. Several single cell cultures were establishedfrom this initial culture. The cultures were maintainedon MV agar butts as stab cultures. The typeculture, on which the description and analyses arebased is deposited at the American Type CultureCollection (ATCC), USA under the accession

ARTICLE IN PRESS312 L. Bongiorni et al.Table 1. Primers used in this study.Primer name Primer sequence Direction Reference18S001 5 0 -AACCTGGTTGATCCTGCCAGTA-3 0 Forward Honda et al. 199918S13 5 0 -CCTTGTTACGACTTCACCTTCCTCT-3 0 Reverse Honda et al. 1999NS3 5 0 -GCAAGTCTGGTGCCAGCAGCC-3 0 Reverse White et al. 1990NS4 5 0 -CTTCCGTCAATTCCTTTAAG-3 0 Forward White et al. 1990M13F 5 0 -GTAAAACGACGGCCAGT-3 0 ForwardM13R 5 0 -AACAGCTATGACCATG-3 0 Reversenumber ATCC PRA-148. A subculture of this isalso deposited in the culture collection of theNational Institute of Oceanography, Goa, underAccession number NIOS-6.Development of the thraustochytrid was studiedusing sea water/pine pollen cultures or 2 days oldcultures in MV broth. A modified design of acontinuous flow micro-chamber as described byRaghukumar (1987) was used for observing thelife cycle under a microscope for up to 3 days. Allobservations were made using an Olympus BX 60microscope and photomicrography using OlympusPM-20 unit.The 18S rRNA gene-based phylogenetic analysiswas undertaken to ascertain the taxonomicaffiliation of the thraustochytrid. Most of themethods used for the purpose were as detailedby Sambrook et al. (1989), except for the indicatedmodifications. Total genomic DNA was isolatedaccording to the procedure of Sheriff et al. (1994).The concentration and purity of the DNA waschecked on 0.8% agarose gel in 1% TAE buffer.The small subunit rRNA gene was amplified bypolymerase chain reaction (PCR) on the DNAthermocycler PTC 200 (MJ Research), using the18S rDNA specific terminal primers 18S001 and18S13 (Table 1). The primers corresponded to thenucleotides 1—22 and 1,733—1,757 of theOchromonas danica 18S rRNA gene (accessionnumber: M32704, J02950) (Honda et al. 1999).Approximately 25 ng of genomic DNA was taken in25 ml reaction volume containing 1 PCR buffer,200 mM each of dATP, dCTP, dGTP and dTTP, 5pMoles of each primer, 1.5 mM MgCl 2 , and 1.0 UampliTaq Gold DNA polymerase (Applied Biosystems).Amplification conditions comprised: aninitial denaturation step of 10 min at 94 1C followedby 35 cycles of I min denaturation at 94 1C, 1 minof annealing at 55 1C and 2 min of extension stepat 72 1C and in the end a final extension step of10 min at 72 1C. The PCR products were checkedon 1.5% agarose gel in 1% TAE buffer beforefurther characterization by cloning and sequencing.The PCR products were ligated into pCR s 2.1vector using the TA cloning kit (Invitrogen) accordingto the instructions of the manufacturer. Theconstructs were transformed into Escherichia coliDH5a competent cells by heat shock method.Recombinant plasmids were isolated by alkalinelysis method and inserts were amplified using M13universal primers (Table 1). Amplified inserts werethen sequenced for both the strands on anautomated DNA sequencer ABI-Prism3700 usingthe fluorescence-based detection approach anddideoxy-termination sequencing chemistry as perthe manufacturer’s details (Applied Biosystems).For sequencing, two additional internal primers(Table 1) were used in conjunction with the M13universal primers that were originally used toamplify the cloned inserts.The 18S rRNA gene sequences were edited andassembled using Auto-assembler program (AppliedBiosystems) and deposited in the NCBInucleotide sequence databank (GenBank AccessionNo. AY705753). The final sequences wereused to identify and retrieve 36 related homologoussequences of reference organisms(Table 2) available in the GenBank database(National Center for Biotechnology Information,USA: NCBI Home page http://www.ncbi.nlm.nih.-gov), using the BLASTn program (Altschul et al.1990). The 18S rDNA sequences obtained for theorganism were aligned with corresponding sequencesbelonging to the 34 reference Labyrinthulomycetes(Table 2) using the programsClustal-X (http://www.igbmc.u-strasbg.fr/BioInfo/)(Higgins and Sharp 1989) and GenDoc (http://www.psc.edu/biomed/genedoc) (Nicholas et al.1997) and also checked manually for large gaps.The aligned sequences were flushed at the endsto avoid missing information for any comparedreference entries. This resulted in a final alignmentof 1600 bp, which was used for further compar-

ARTICLE IN PRESSA new Thraustochytrid from Mangroves 313Table 2. List of 18S rRNA gene sequences of relatedLabyrinthulomycota species used as reference taxain this study for ascertaining the generic affiliation ofthe new isolate.OrganismGenBankaccessionnumberOxytricha granuliferaX53486Prorocentrum micansM14649Aplanochytrium kerguelense AB 022103Japanochytrium Sp.AB022104Labyrinthuloides haliotidisU21338Labyrinthula Sp.AB022105Schizochytrium aggregatum AB022106Schizochytrium limaciniumAB022107Schizochytrium minutumAB022108Thraustochytrium aggregatum AB022109Thraustochytrium aureumAB022110Thraustochytrium kinneiL34668Thraustochytrium multirudimentale AB022111Thraustochytrium striatumAB022112Thraustochytrium pachydermum AB022113Ulkenia profundaL34054Ulkenia profunda (#29)AB022114Ulkenia radiataAB022115Ulkenia visurgensisAB022116Thraustochytriidae sp. N4-103 AB073309Thraustochytriidae sp. N1-27 AB073308Thraustochytriidae sp. H6-16 AB073306Thraustochytriidae sp. H1-14 AB073305Thraustochytriidae sp. F3-1AB073304Thraustochytrium sp. CHN-1 AB126669Aplanochytrium stocchinoiAJ519935Schizochytrium sp. KH105AB052555Thraustochytrium sp. KK17-3 AB052556Labyrinthulid quahog parasite QPX AF261664Labyrinthulid quahog parasite QPX AF155209Aplanochytrium sp. SC1-1AF348520Aplanochytrium sp. PR1-1AF348516Aplanochytrium sp. PR12-3AF348517Aplanochytrium sp. PR15-1AF348518Labyrinthula sp.AF348522Thraustochytriidae sp. M4-103 AB073307isons. The aligned sequences were then used toderive corrected Kimura two-parameter distance(Kimura 1980) estimates and infer phylogeneticrelationships using both distance based as well asdirect character-state-based methods, namely,neighbour-joining (Saitou et al. 1987) and maximumlikelihood, with analytical routines availablein the software packages PHYLIP 3.5c (Felsenstein1993) (http://evolution.genetics.washington.edu/phylip.html) and Phylo_win (Galtier et al.1996). In phylogenetic analysis, the two alveolates,Prorocentrum micans (Dinoflagellata) andOxytricha granulifera (Ciliphora) were selected asoutgroups to root the phenetic trees as they oftenform sister groups with the straminopiles in the18S rDNA eukaryotic tree. The robustness of thephenetic clustering was ascertained by 500 bootstrapresamplings (Felsenstein 1985).AcknowledgementsOne of us (L.B.) is grateful to the Indian Council ofCultural Relations, New Delhi for the award of aPost-doctoral fellowship under the Indo-ItalianCultural Exchange Programme, during the tenureof which this work was carried out. We are alsothankful to the Council of Scientific and IndustrialResearch, New Delhi and the Directors of NIO andCCMB for support. This is NIO contribution no.4003ReferencesAinsworth GC, Sparrow FK Jr., Sussman AS eds(1973) The Fungi. An Advanced Treatise, Vol. IV B.Academic Press, New YorkAltschul SF, Gish W, Miller W, Myers EW, LipmanDJ (1990) Basic local alignment search tool. J MolBiol 215: 403—410Cavalier-Smith T (1993) Kingdom Protozoa and its18 phyla. Microbiol Rev 57: 953—994Cavalier-Smith T, Allsop M, Chao EE (1994)Thraustochytrids are chromists not fungi: signaturesequences of Heterokonta. Philos Trans R Soc LondB 346: 387—397Chamberlain AHL (1980) Cytochemical and ultrastructuralstudies on the cell walls of Thraustochytriumspp Bot Mar 23: 669—677Chamberlain AHL, Moss ST (1988) The thraustochytrids:a protist group with mixed affinities.Biosystems (Amsterdam) 21: 341—349Darley WM, Porter D, Fuller M (1973) Cell wallcomposition and synthesis via Golgi-directed scaleformation in the marine eucaryote, Schizochytriumaggregatum, with a note on Thraustochytrium sp.Arch Mikrobiol 90: 89—106Dick MW (2001) Straminipilous Fungi. Kluwer AcademicPublishers, The Netherlands

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