Mycoscience (2007) 48:199–211 DOI 10.1007/s10267-006-0362-0 © The Mycological Society of Japan and Springer 2007 FULL PAPER Rinka Yokoyama · Daiske Honda Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov. Received: November 25, 2006 / Accepted: December 13, 2006 Abstract The genus Schizochytrium sensu lato has been characterized by successive binary division of its vegetative cells. However, the molecular phylogeny strongly suggests that this genus is not a natural taxon, because the original and recorded strains that have been identified as Schizochytrium spp. separately form three well-supported monophyletic groups in the 18S rRNA gene tree. These three groups are clearly distinguishable by their combined morphological characteristics and the profiles of the polyunsaturated fatty acids and carotenoid pigments they contain, although these are hard to distinguish using only a single feature. Therefore, three different genera are proposed to accommodate these three groups, i.e., Schizochytrium sensu stricto, Aurantiochytrium, and Oblongichytrium gen. nov. Key words Carotenoids · Heterokonts · Polyunsaturated fatty acids (PUFAs) · Thraustochytrium · Ultrastructure Introduction The class Labyrinthulomycetes (Levine and Corliss 1963; Olive 1975; Levine et al. 1980) is a member of the heterotrophic stramenopiles, which are characterized and distinguished from other fungoid organisms by the following characteristics: biflagellate zoospores possessing an anterior flagellum with mastigonemes (Amon and Perkins 1968; Kazama 1974; Perkins 1974), rhizoid-like ectoplasmic net elements produced by a unique organelle, the bothrosome (= sagenogen, sagenogenetosome) (Perkins 1972; Porter 1972, 1974; Moss 1980, 1985), and multilamellate cell walls R. Yokoyama Graduate School of Natural Science, Konan University, Hyogo, Japan D. Honda (*) Department of Biology, Faculty of Science and Engineering, Konan University, 8-9-1 Okamoto, Higashinada, Kobe, Hyogo 658-8501, Japan Tel. +81-78-435-2515; Fax +81-78-435-2539 e-mail: dhonda@konan-u.ac.jp composed of Golgi body-derived scales (Alderman et al. 1974; Perkins 1974; Porter 1974; Moss 1985). This class is composed of two families (Olive 1975; Porter 1989): Thraustochytriaceae Sparrow ex Cejp (Cejp 1959; see also Sparrow 1943, 1960), characterized by globose cells with ectoplasmic nets from a single bothrosome, and Labyrinthulaceae Cienk. (Cienkowski 1867; see also Haeckel 1868), having spindleshaped cells gliding through channels of ectoplasmic nets produced from a number of bothrosomes. Classification of six genera in the Thraustochytriaceae was based on the cell morphology of various stages during the life cycle. The type genus of this family is Thraustochytrium Sparrow emend. T.W. Johnson, which has been characterized by its globose sporangia with or without proliferous bodies and zoospore release caused by partial dissolution of the cell wall of the sporangia (Sparrow 1936; Johnson and Sparrow 1961). The other genera have subsequently been separated or newly erected on the basis of morphological features as follows: Japonochytrium Kobayasi et M. Ookubo is distinguished by the apophysis at its ectoplasmic nets (Kobayashi and Ookubo 1953); Schizochytrium S. Goldst. et Belsky emend. T. Booth et C.E. Mill. (i.e., Schizochytrium sensu lato) undergoes multiplication by binary cell division of vegetative cells (Goldstein and Belsky 1964); Althornia E.B.G. Jones et Alderman lacks the bothrosome and ectoplasmic nets (Jones and Alderman 1971); Ulkenia A. Gaertn. releases amoeboid cells before forming sporangia (Gaertner 1977); and Aplanochytrium Bahnweg et Sparrow emend. C.A. Leander et D. Porter is characterized by release of aplanospores and motility of vegetative cells (Bahnweg and Sparrow 1972; Leander and Porter 2000). However, the genus-level classification has been problematical. The characteristic features overlap among some species of the genus Thraustochytrium (Booth and Miller 1968; Sparrow 1969; Alderman et al. 1974). It has been suggested that Thraustochytrium is a permissive and unarranged group, including morphologically diversified species (cf. Karling 1981). Also, amoeboid cells have been observed not only in Ulkenia but also in Schizochytrium sensu lato and in Thraustochytrium species under some culture conditions, and there is therefore some dispute as to the validity 200 of the diagnostic characteristics for the genus classification (Raghukumar 1988a; Honda et al. 1998; Bongiorni et al. 2005). Moreover, Honda et al. (1999) clearly showed that none of these three genera formed a monophyletic group in the molecular phylogenetic tree of 18S rRNA genes, and it was therefore assumed by them that these genera were not natural taxa. Consequently, the currently used taxonomic criteria might need serious reconsideration, and the classification should be rearranged, if necessary. One of the problematical genera, Schizochytrium sensu lato, is composed of the following five species: S. aggregatum S. Goldst. et Belsky, the type species of the genus, is characterized by the formation of large clusters of cells (Goldstein and Belsky 1964); S. minutum A. Gaertn. releases only two zoospores from one sporangium (Gaertner 1981); S. octosporum Raghuk. releases eight zoospores from one sporangium (Raghukumar 1988b); S. mangrovei Raghuk. does not form a zoosporangium, and each vegetative cell develops into zoospores (Raghukumar 1988a); and S. limacinum D. Honda et Yokochi is characterized by 16– 32 clustered cells and amoeboid cells (Honda et al. 1998). However, in an 18S rRNA gene sequence tree, at least three species, S. aggregatum, S. minutum, and S. limacinum, were located in three independent lineages (Honda et al. 1999). This result means that the characteristic feature, that is, successive binary division of the vegetative cells, has been gained in several lineages. Conversely, it can be interpreted that the loss of vegetative cell division occurred in many thraustochytrid lineages classified as different genera, because the deeply branched groups, Aplanochytrium and Labyrinthula Cienk., also possess this feature (Leander and Porter 2001). It will therefore be necessary to assess how many phylogenetic lineages in the thraustochytrids with the ability of vegetative cell division are identified as Schizochytrium sensu lato. The production of polyunsaturated fatty acids (PUFAs) by thraustochytrids, especially docosahexaenoic acid (DHA, C22:6, n-3), has recognized commercial use (Bowles et al. 1999; Lewis et al. 1999). Huang et al. (2003) showed that the PUFA profiles of strains in the monophyletic groups in the 18S rRNA gene tree were fundamentally similar and that each monophyletic group could be distinguished by its PUFA profile. The production of carotenoid pigments of the thraustochytrids has also received attention from the industry (Valadon 1976; Aki et al. 2003; Carmona et al. 2003; Yamaoka et al. 2004). These studies suggest that profiles of carotenoid pigments differ according to individual thraustochytrid strains. In the present study, the following strategies were accomplished. As the first step, thraustochytrids were randomly collected from the field, and objective strains with binarydivided vegetative cells were selected. As the second step, the molecular phylogenetic positions of the strains were revealed by analyses of the sequences of their 18S rRNA gene. As the third step, selected strains were examined morphologically by light and electron microscopy, PUFAs, and carotenoid pigments. Finally, comparison of the entire set of data resulted in a general discussion on taxonomic rearrangement. Materials and methods Samples and cultivation The examined strains and their sources are shown in Table 1. The original strains, shown by the prefix SEK, were isolated from seawater and mud collected at each site of Japan by pine-pollen baiting methods (Gaertner 1968). Cells were cultured at 25°C in medium-H (Honda et al. 1998). Observation Cells of each stage in the life cycle were cultured in both medium-H and seawater/pine-pollen cultures, which were proposed by Raghukumar (1988a) as a standard medium for identification. Light microscopic observations were by an BX60 (Olympus, Tokyo, Japan) fitted with a Nomarski interference differential contrast objective. These images were captured with an AxioCam HRc digital camera controlled by AxioVision software release 4.4 under the normal setting with no digital gain (Carl Zeiss, Hallbergmoos, Germany). For continuous observation, cells were transferred into a glass-bottomed culture dish (Meridian Instruments Far East, Tokyo, Japan) filled with fresh medium-H or seawater/pine pollen cultures. Cells started to develop after cultivation for 3–4 h and were observed with a CK40 (Olympus) or Axiovert 200 (Carl Zeiss) inverted microscope with a CCD camera CS900 (Olympus). Images were captured at one frame/2 s by Moto DV (Digital Origin, Palo Alto, CA, USA) on a Power Mac G4 (Apple Computer, Cupertino, CA, USA). Vegetative cells were fixed in 2.5% glutaraldehyde and 0.1% sucrose in 0.1 M cacodylate buffer on ice for 5 h. After rinsing with the same buffer, materials were subsequently fixed in 1% OsO4 for 12 h on ice, followed by washing in the buffer. The specimens were dehydrated in 30%, 50%, 70%, 90%, 95%, and 100% ethanol for 10 min each, followed by both ethanol-propylene oxide (PO) mixtures and pure PO twice for 10 min. Next, the specimens were embedded in pure epoxy resin (Quetol-651; Nisshin EM, Tokyo, Japan). The chamber was then placed in a 60°C oven where the resin was allowed to polymerize. Ultrathin sections were cut on a microtome (Leica Ultracut R; Leica, Wien, Austria) and were stained for 15 min with 4% uranyl acetate, followed by 5 min in Reynolds’ lead citrate (Reynolds 1963). The sections were viewed under a JEM 2000FX (JEOL, Tokyo, Japan) transmission electron microscope. Extraction of DNA Cells were harvested after 3–4 days of culture. Total genomic DNA was extracted using the method suggested by the instructions in the Genomic Prep Cells and Tissue DNA Isolation Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and was purified using a phenol and chloroform/ isoamyl alcohol protocol. To obtain the almost complete 18S rRNA gene, we used a polymerase chain reaction (PCR) 201 Table 1. The strains used in the molecular phylogenetic analyses with sequence accession numbers Taxon Genus Aurantiochytrium Aurantiochytrium limacinum Aurantiochytrium mangrovei Aurantiochytrium sp. Aurantiochytrium sp. Aurantiochytrium sp. Aurantiochytrium sp. Aurantiochytrium sp. Aurantiochytrium sp. Aurantiochytrium sp. Aurantiochytrium sp. Genus Oblongichytrium Oblongichytrium minutum Oblongichytrium multirudimentale Oblongichytrium sp. Oblongichytrium sp. Oblongichytrium sp. Genus Schizochytrium Schizochytrium aggregatum Schizochytrium sp. Schizochytrium sp. Schizochytrium sp. Schizochytrium sp. Schizochytrium sp. Other strains Aplanochytrium kerguelense Aplanochytrium minutum Aplanochytrium stocchinoi Aplanochytrium sp. Aplanochytrium sp. Aplanochytrium sp. Japonochytrium sp. Labyrinthula sp. Labyrinthula sp. Labyrinthula sp. Labyrinthula sp. Thraustochytrium agggregatum Thraustochytrium aureum Thraustochytrium kinnei Thraustochytrium pachydermum Thraustochytrium striatum Ulkenia profunda Ulkenia profunda Ulkenia radiata Ulkenia visurgensis Outgroup Bacillaria paxillifer Ochromonas danica Straina Accession number References NIBH SR21T (IFO 32693) RCC893 BURABQ 133 SEK 217 (NBRC 103268) SEK 218 (NBRC 103269) SEK 209 (NBRC 102614) NIOS-4 FJN-10 NIBH N1-27 KH105 Uncultured thraustochytrid AB022107 DQ367049 DQ023620 AB290572 AB290573 AB290574 AY705751 AY773276 AB073308 AB052555 DQ023610 Honda et al. 1999 – – This study This study This study – – Huang et al. 2003 Huang et al. 2003 – KMPB N-BA-77T KMPB N-BA-113 SEK 347 (NBRC 102618) 7-5 8-7 AB022108 AB022111 AB290575 AF257316 AF257317 Honda et al. 1999 Honda et al. 1999 This study Mo et al. 2002 Mo et al. 2002 ATCC 28209 SEK 210 (NBRC 102615) SEK 345 (NBRC 102616) SEK 346 (NBRC 102617) KK17-3 NIBH N4-103 AB022106 AB290576 AB290577 AB290578 AB052556 AB073309 Honda et al. 1999 This study This study This study Huang et al. 2003 Huang et al. 2003 KMPB N-BA-107 n/a PR1-1 PR15-1 SC1-1 ATCC 28207 AN-1565 (NBRC 33215) L59 L72 f Sap 16-1 KMPB N-BA-110T ATCC 34304T KMPB 1694d KMPB N-BA-114 ATCC 24473T #29 (Raghukumar) KMPB N 3077T #16 (Raghukumar) ATCC 28208T NIBH H1-14 BS1 BS2 NIOS-6 (A05-2) C9G Fug1 QPX AB022103 L27634 AJ519935 AF348516 AF348518 AF348520 AB022104 AB022105 AB095092 AB220158 AF348522 AB022109 AB022110 L34668 AB022113 AB022112 AB022114 L34054 AB022115 AB022116 AB073305 AF257314 AF257315 AY705756 AF474172 AY870336 AY052644 Honda et al. 1999 Leipe et al. 1994 Moro et al. 2003 Leander et al. 2004 Leander et al. 2004 Leander et al. 2004 Honda et al. 1999 Honda et al. 1999 Kumon et al. 2003 Kumon et al. 2006 – Honda et al. 1999 Honda et al. 1999 Cavalier-Smith et al. 1994 Honda et al. 1999 Honda et al. 1999 Honda et al. 1999 Cavalier-Smith et al. 1994 Honda et al. 1999 Honda et al. 1999 Huang et al. 2003 Mo et al. 2002 Mo et al. 2002 – Anderson et al. 2003 – Stokes et al. 2002 n/a n/a M87325 M32704, J02950 – – a Abbreviations of the culture collections; ATCC, American Type Culture Collection; IFO, Institute for Fermentation, Osaka (Japan); KMPB, Kulturensammlung Mariner Pilze Bremerhaven, Alfred-Wegner-Institut für Polar und Meeresforschung (Germany); NBRC, NITE (National Institute of Technology and Evaluation)- Biological Resource Center (Japan); NIBH, National Institute of Bioscience and Human Technology (Japan); SEK, Laboratory of Systematics and Evolution at Konan University (Japan) T, ex-type strain; n/a, not available protocol with a thermostable DNA polymerase (Ex Taq DNA Polymerase; Takara, Ohtsu, Japan) and amplification primers SR1 and SR12 (Nakayama et al. 1996). We cut out the amplified band of about 1800 bases in 0.5% TBE-agarose gel and then extracted it using Gene Clean Turbo (Qbio- gene, Baton Rouge, LA, USA). Nucleotide sequences were determined by the terminator method with 12 primers, 18S01–18S12, the same as those used by Nakayama et al. (1996), using an ABI PRISM 310NT Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). 202 Molecular phylogenetic analyses Determined sequences were added to the aligned sequence data set (Honda et al. 1999) through a profile alignment process using Clustal W version 1.74 (Thompson et al. 1994) and optically aligned. Other strain sequences were downloaded from the GenBank constructed by the National Center of Biotechnology Information (NCBI) (accession numbers are shown in Table 1). The phylogenetic trees were generated using the maximum-likelihood (ML) method (Felsenstein 1981) and the minimum-evolution (ME) method (Rzhetsky and Nei 1992, 1993). Both analyses were performed using PAUP* version 4.0b10 (Swofford 2003). In ML method analysis, the transition/transversion (ti/tv), the ratio of the HKY85 model, was estimated by maximizing the likelihood value for neighbor-joining (NJ) topology. The best tree was found by the heuristic search method. The bootstrap values were obtained from 100 resamplings. In the distance method analysis, the total distance of a tree was calculated using the ML parameter based on the ti/tv ratio that was estimated from NJ topology. The best tree was found by heuristic search. The bootstrap values were obtained from 1000 resamplings. PUFA composition analyses Each strain of our own isolates was previously cultured in medium-H for 2–3 days at 25°C. The cell suspension was spread on the agar-dGPY medium containing 2 g glucose, 1 g poly-peptone, 0.5 g yeast extract, and 15 g agar per liter of a half-salt concentration of artificial seawater and then incubated for 7–10 days at 25°C. The cell growths were gathered by scraping a small spatula across the surface and dried at 105°C for 3 h. The fatty acids were directly transmethylated from dried cells with 10% methanolic HCl and methylene chloride (Shimizu et al. 1988). The methyl-esterified fatty acids were extracted with n-hexane, and the resultant extracts were applied to a gas/liquid chromatograph (GC-17A; Shimadzu, Kyoto, Japan) equipped with a TC-70 capillary column (25 m × 0.25 mm i.d.; GL Science, Tokyo, Japan); a temperature program rising from 180° to 220°C in increments of 4°C/min was used. Peaks were identified using authentic standards of the following fatty acid methyl esters according to Nakahara et al. (1996) and Yokochi et al. (1998): arachidonic acid (AA, 20:4 n-6), eicosapentaenoic acid (EPA, 20:5 n-3), docosapentaenoic acid (DPA, 22:5 n-3 and n-6), and docosahexaenoic acid (DHA, 22:6 n-3). Each PUFA production was calculated from each peak area of the chromatogram relative to the peak area of an internal standard. Carotenoid composition analyses Each strain was inoculated into a 500-ml conical flask containing 200 ml medium-H and then incubated with reciprocal shaking at 120 rpm for 2–3 days at 25°C. The growth cells were harvested by centrifugation at 2500 rpm for 15 min. The pellets were freeze-dried on liquid nitrogen. The endopigments were extracted by suspending the cells in 1 ml acetone and crushing in a mortar, after which the pigment solutions were filtrated through an EkicrodiscR 3 CR filter (0.45 µm × 3 mm; Gelman Science, Tokyo, Japan). The resulting solutions were loaded into a HPLC equipped with a UV-VIS detector (SPD6AV; Shimazu, Kyoto, Japan), which was set at 450 nm. Astaxanthin, phoenicoxanthin, canthaxanthin, echinenone, and β-carotene were identified using the peaks of the standard sample and the data of previous studies (Carmona et al. 2003). Taxonomy According to comparison of all the data sets shown later, the following three genera, including two new genera, are recharacterized or established in the Thraustochytriaceae, Labyrinthulales, and Labyrinthulomycetes. Schizochytrium S. Goldst. et Belsky emend. R. Yokoyama et D. Honda Thallus thin-walled, globose, pale yellow. Cells possessing only β-carotene as carotenoid pigment and possessing ca. 20% arachidonic acid as the fatty acids. Colonies large by continuous binary cell divisions. Ectoplasmic nets well developed. Zoospores biflagellate heterokont; reniform to ovoid. 18S rRNA gene sequence distinct. Resting spores not observed. Type species: Schizochytrium aggregatum S. Goldst. et Belsky Oblongichytrium R. Yokoyama et D. Honda, gen. nov. Thallus pariete tenui, globosus, lutescens. Cellulae cum pigmentis dictis “Canthaxanthin,” “β-carotene,” et cum acido pingui comparate abundanto “n-3 docosapentaenoic acid” dicto et pauco “n-6 docosapentaenoic acid” dicto. Coloniae comparate evolventes per fissionem binariam cellulae diadosae. Reticulum ectoplasmaticum conspicue evolvens. Zoosporae ellipticae vel oblongae, biflagellatae, heterokontae. 18S rRNA genetica distincta. Sporae perdurantes non observatae. Thallus thin-walled, globose, pale yellow. Cells possessing canthaxanthin, β-carotene, and possessing comparatively abundant n-3 docosapentaenoic acid and little n-6 docosapentaenoic acid as the fatty acids. Colonies large by continuous binary cell divisions. Ectoplasmic nets well developed. Zoospores biflagellate heterokont; elliptical to oblong. 18S rRNA gene sequence distinct. Resting spores not observed. Etymology: oblongus = oblong, chytrion = pot, referring the oblong zoospores. Type species: Oblongichytrium minutum (A. Gaertn.) R. Yokoyama et D. Honda, comb. nov. Basionym: Schizochytrium minutum A. Gaertn., Veröff Inst Meeresforsch Bremerh 19:68, 1981. 203 Oblongichytrium multirudimentale (S. Goldst.) R. Yokoyama et D. Honda, comb. nov. Basionym: Thraustochytrium multirudimentale S. Goldst., Am. J. Bot. 50:273, 1963. Other species that we believe belong to Oblongichytrium: Oblongichytrium octosporum (Raghuk.) R. Yokoyama et D. Honda, comb. nov. Basionym: Schizochytrium octosporum Raghuk, Trans. Br. Mycol. Soc. 90:273, 1988. Aurantiochytrium R. Yokoyama et D. Honda, gen. nov. Thallus pariete tenui, globosus, aurantiacus. Cellulae cum pigmentis dictis “astaxanthin,” “phoenicoxanthin,” “canthaxanthin,” “β-carotene,” et cum acido pingui pauco “arachidonic acid” dicto, et cum acido pingui dominanto “docosahexaenoic acid” dicto. Coloniae comparate parvae per fissionem binariam cellulae diadosae. Reticulum ectoplasmaticum non evolvens comparate. Zoosporae reniformes vel ovoldeae, biflagellatae, heterokontae. 18S rRNA genetica distincta. Sporae perdurantes non observatae. Thallus thin-walled, globose, orange. Cells possessing astaxanthin phoenicoxanthin, canthaxanthin, and βcarotene and possessing minor arachidonic acid and dominant docosahexaenoic acid as the fatty acids. Colonies small by continuous binary cell divisions. Ectoplasmic nets not well developed. Zoospores biflagellate heterokont; reniform to ovoid. 18S rRNA gene sequence distinct. Resting spores not observed. Etymology: aurantius = orange in color, chytrion = pot, referring the color of the thallus. Type species: Aurantiochytrium limacinum (D. Honda et Yokochi) R. Yokoyama et D. Honda, comb. nov. Basionym: Schizochytrium limacinum D. Honda et Yokochi in Honda et al. Mycol. Res. 102:441, 1998. Aurantiochytrium mangrovei (Raghuk.) R. Yokoyama et D. Honda, comb. nov. Basionym: Schizochytrium mangrovei Raghuk, Trans. Br. Mycol. Soc. 90:627, 1998. Results Light microscopic morphology Diads and tetrads were observed in the life cycles of all studied isolates (Fig. 1). A settled zoospore transformed into some sporangia through binary cell division. This type of multiplication had previously been characterized as belonging to the genus Schizochytrium sensu lato (Goldstein and Belsky 1964). Molecular phylogeny of 18S rRNA gene sequences Almost the entire length of the 18S rRNA gene sequences was determined and deposited in the DNA Data Bank of Japan (DDBJ) (see Table 1), and the alignment was deposited in TreeBASE (matrix accession number: M3311 at a d g b e c f h Fig. 1. Light micrographs showing cluster of cells via binary cell division on the medium-H. a Aurantiochytrium limacinum; b Aurantiochytrium sp. SEK 209; c Aurantiochytrium sp. SEK 217; d Schizochytrium aggregatum; e Schizochytrium sp. SEK 210; f Schizochytrium sp. SEK 326; g Oblongichytrium sp. SEK 347; h Oblongichytrium sp. SEK 347. Bars 5 µm http://treebase.org/). We used 1235 sites for comparison among all operational taxonomic units without the gaps and ambiguous sites. The strains of Schizochytrium sensu lato separated into three independent monophyletic groups (Fig. 2). The clade of Aurantiochytrium diverged with a long branch from the internal node, including strains of A. limacinum NIBC SR21, Aurantiochytrium sp. SEK 209, Aurantiochytrium sp. SEK 217, and Aurantiochytrium sp. SEK 218, and was supported by 100% bootstrap values in both ML and ME analyses. The clade of Schizochytrium sensu stricto, including strains of S. aggregatum ATCC 28209, Schizochytrium sp. SEK 210, Schizochytrium sp. SEK 345, and Schizochytrium sp. SEK 346, was also supported by 100% bootstrap values in both analyses. The clade of Oblongichytrium, including strains of O. minutum, O. multirudimentale, and Oblongichytrium sp. SEK 347, was supported by 90% and 100% bootstrap values in ML and ME analyses, respectively. The members of the genus Labyrinthula formed a monophyletic group supported by 100% bootstrap values in both analyses. Members of the genus Aplanochytrium 204 Aurantiochytrium limacinum Aurantiochytrium sp. BURABQ_133 100 Aurantiochytrium mangrovei uncultured thraustochytrid 100 92 Aurantiochytrium sp. SEK218 100 Aurantiochytrium sp. SEK217 Aurantiochytrium sp. NIOS_4 Aurantiochytrium sp. KH105 100 Aurantiochytrium sp. SEK209 Aurantiochytrium sp. N1_27 93 Aurantiochytrium sp. FJN_10 29 Thraustochytrium aureum 51 Thraustochytrium kinnei 100 Ulkenia profunda_29 43 43 Ulkenia radiata 70 H1_14 Thraustochytrium striatum 100 Japonochytrium sp. 100 Ulkenia visurgensis 73 34 Ulkenia profunda_KMPB Schizochytrium aggregatum Schizochytrium sp. SEK346 90 99 Schizochytrium sp. SEK210 20 100 Schizochytrium sp. SEK345 77 Schizochytrium sp. KK17_3 96 99 Schizochytrium sp. N4_103 NIOS_6 Fug1 100 BS2 29 BS1 Thraustochytrium aggregatum 88 Thraustochytrium pachydermum 40 47 C9G 96 QPX Aplanochytrium kerguelense Aplanochytrium minutum 88 Aplanochytrium sp. SC1_1 Aplanochytrium sp. PR1_1 87 Aplanochytrium sp. PR15_1 100 Aplanochytrium stocchinoi 91 Labyrinthula sp. 100 Labyrinthula sp. L59 100 Labyrinthula sp. L72 99 Labyrinthula sp. f_sap 59 Oblongichytrium minutum Oblongichytrium multirudimentale Oblongichytrium sp. SEK347 90 83 Oblongichytrium sp. 7_5 Oblongichytrium sp. 8_7 Bacillaria paxillifer Ochromonas danica a (ML) 65 100 Aurantiochytrium Schizochytrium sensu stricto Oblongichytrium 0.05 substitutions/site Aurantiochytrium limacinum Aurantiochytrium mangrovei uncultured thraustochytrid Aurantiochytrium sp. BURABQ_133 100 Aurantiochytrium sp. SEK218 100 Aurantiochytrium sp. SEK217 Aurantiochytrium sp. NIOS_4 Aurantiochytrium sp. KH105 99 Aurantiochytrium sp. SEK209 Aurantiochytrium sp. FJN_10 100 Aurantiochytrium sp. N1_27 Thraustochytrium aureum 76 50 100 Ulkenia profunda_29 Ulkenia radiata H1_14 74 Thraustochytrium striatum 27 100 Japonochytrium sp. 100 Ulkenia visurgensis Ulkenia profunda_KMPB Schizochytrium aggregatum Schizochytrium sp. SEK346 95 71 100 Schizochytrium sp. SEK210 100 Schizochytrium sp. SEK345 69 Schizochytrium sp. KK17_3 94 Schizochytrium sp. N4_103 NIOS_6 36 Thraustochytrium kinnei BS2 100 BS1 77 Thraustochytrium pachydermum 100 C9G 100 QPX Fug1 Thraustochytrium aggregatum Aplanochytrium kerguelense Aplanochytrium minutum 100 Aplanochytrium sp. SC1_1 Aplanochytrium stocchinoi Aplanochytrium sp. PR15_1 73 Aplanochytrium sp. PR1_1 94 Oblongichytrium minutum Oblongichytrium multirudimentale 100 79 Oblongichytrium sp. SEK347 Oblongichytrium sp. 7_5 100 Oblongichytrium sp. 8_7 Labyrinthula sp. 100 Labyrinthula sp. L59 100 100 Labyrinthula sp. L72 Labyrinthula sp. f_sap Bacillaria paxillifer Ochromonas danica b (ME) 100 59 90 100 100 Aurantiochytrium Schizochytrium sensu stricto Oblongichytrium 66 0.05 substitutions/site Fig. 2. Phylogenetic trees of the Labyrinthulomycetes using 18S rRNA gene with Bacillaria paxillifer and Ochromonas danica as outgroup (51 operational taxonomic units, 1235 nucleotide sites). a The best maximum-likelihood (ML) tree (log-likelihood = –133766.32608) on the HKY85 model (ti/tv ratio = 1.071671). b The best minimum-evolution (ME) tree constructed from distances estimated by the ML method with HKY85 model (ti/tv ratio = 1.071671). The numbers at each internal branch show the bootstrap values (%) for the nodes calculated by 100 and 1000 replicates on ML and ME analyses, respectively. Bold characters indicate our original isolates, whose sequences were determined in this study also formed a monophyletic group and made a sister relationship with the clade of Labyrinthula. The clade of Oblongichytrium spp. branched out deeply from the basal node near Labyrinthula and Aplanochytrium clades. sp. SEK 217, 2.0–3.0 × 4.0–5.0 µm in Fig. 3d–f). Under ultrastructural observations, there were electron-dense bodies (EDB) around the nuclei of all strains (Fig. 3g–i). Paranuclear bodies (PA) were also observed (Fig. 3j–l). A PA composed of a complex of inflated smooth endoplasmic reticulum cisternae has been previously demonstrated in other genera, namely, Aplanochytrium yorkense (F.O. Perkins) C.A. Leander et D. Porter (Perkins 1973), Ulkenia visurgensis Ulken emend. A. Gaertner (Moss 1980, 1985), and Ulkenia amoeboidea Bahnweg et Sparrow (Raghukumar 1982a,b). A typical vegetative cell has a nucleus, a vacuole, a Golgi body, and mitochondria with tubular cristae (Fig. 3l). The vegetative cells of Schizochytrium sensu stricto (S. aggregatum ATCC 28209, Schizochytrium sp. SEK 210, Schizochytrium sp. SEK 346) form large colonies with well-developed ectoplasmic nets in both seawater/pine pollen culture and enriched media (Fig. 4a–c). The zoospores are ovoid in shape, similar to those of the Schizochytrium sensu lato strains (S. aggregatum ATCC 28209, 2.5–3.0 × 4.0–7.5 µm; Schizochytrium sp. SEK 210, 2.5–3.0 × Morphological characteristics Morphological features are summarized in Table 2. Colonies of the vegetative cells of Aurantiochytrium (A. limacinum NIBH SR21, Aurantiochytrium sp. SEK 209, Aurantiochytrium sp. SEK 217) are dispersed and do not form large colonies. Their ectoplasmic nets are undeveloped in both nutrient-enriched media and seawater/pine pollen culture (Fig. 3a–c). Vegetative cells do not form large colonies and are mostly dispersed as single cells in these liquid media, but occasionally old vegetative cells and mature sporangia do not separate and form clusters with ectoplasmic nets. The zoospores are ovoid in shape (A. limacinum NIBH SR21, 5.0–7.0 × 6.0–8.5 µm; Aurantiochytrium sp. SEK 209, 2.5–3.0 × 4.0–6.0 µm; Aurantiochytrium Large Well developed 3.5–4 × 4.5–6.5 Ovoid 16–64 + − + + Large Well developed 3.5–5 × 7–8.5 Narrow, elliptical 4–67 + − + + 4.0–5.0 µm; Schizochytrium sp. SEK 346, 3.5–4.0 × 4.5– 6.5 µm in Fig. 4d–f). Under ultrastructural examination, EDB (Fig. 4g–i) and PA (Fig. 4j–l) were also observed. A typical vegetative cell has a nucleus, a vacuole, a Golgi body, kinetosome, and mitochondria with tubular cristae (Fig. 4m). The vegetative cells of Oblongichytrium sp. SEK 347 form large colonies with more well-developed ectoplasmic nets compared to those of Aurantiochytrium strains in both seawater/pine pollen culture and enriched media (Fig. 5a). The zoospores are a narrow elliptical shape (3.0 × 7.0 µm; Fig. 5b) and are only released when their sporangia are transferred from the agar cultivation to liquid media. Under ultrastructural observations, EDB (Fig. 5c) and PA (Fig. 5d) were observed in vegetative cells. A typical vegetative cell has a nucleus, mitochondria with tubular cristae, vacuoles, and a Golgi body (Fig. 5d). PUFA and carotenoid composition The PUFA profiles of arachidonic acid (AA, C20:4, n-6), eicosapentaenoic acid (EPA, C20:5, n-6), docosapentaenoic acid (DPA, C22:5, n-3 and n-6), and docosahexanoic acid (DHA, C22:6, n-3) were compared in three genera and other strains including isolates that have reported in Huang et al. (2003). Figure 6 shows that all examined strains of Aurantiochytrium contained less than 5% AA and about 80% DHA, whereas all the Schizochytrium sensu stricto strains contained about 20% AA. Oblongichytrium sp. SEK 347 accumulated about 20% of n-3 DPA, which is unique among the thraustochytrid strains examined. The ketocarotenoids profiles of β-carotene, echinenone, canthaxanthin, phoenicoxanthin, and astaxanthin were compared (Table 3). These pigments are intermediates of the synthetic pathway of astaxanthin from β-carotene. The strains of Aurantiochytrium possess all the aforementioned pigments, resulting in orange colonies. Because the strains of Schizochytrium sensu stricto only contain β-carotene, the colonies are light yellow. Oblongichytrium sp. SEK 347 accumulates canthaxanthin and β-carotene, but not astaxanthin and phoenicoxanthin. The other strains showed different carotenoid profiles; that is, Japonochytrium sp. possesses only astaxanthin and β-carotene, whereas Thraustochytrium striatum Joa. Schneid. contains all five pigments. Discussion ud, undetermined Colony Ectoplasmic nets Size of a zoospore (µm) Shape of zoospore Number of releasing zoospores Cell wall remains after zoospore release Amoeboid cells Electron-dense body Para-nuclear body Small Slightly developed 5–7 × 6–8.5 Ovoid 8–32 − + ud ud Small Undeveloped 2.5–3 × 4–6 Ovoid 8–32 − − + + Small Undeveloped 2–3 × 4–5 Ovoid 8–16 + − + + Large Well developed 2.5–3 × 4–7.5 Ovoid 16–64 + − + + Large Well developed 2.5–3 × 4–5 Ovoid 16–64 + − + + O. sp. SEK347 S. sp. SEK346 S. sp. SEK210 S. aggregatum A. limacinum A. sp. SEK209 A. sp. SEK217 Genus Schizochytrium Genus Aurantiochytrium Table 2. Morphological features of Aurantiochytrium, Oblongichytrium, and Schizochytrium sensu stricto strains Genus Oblongichytrium 205 The genus Schizochytrium sensu lato was characterized by the successive binary divisions of the vegetative cells (Goldstein and Belsky 1964). This characteristic is easily recognized by continuous observation and colony morphology. This characteristic feature also is observed without regard to growth media, whereas the other morphological features under light microscopy are influenced by media (Booth and Miller 1968; Goldstein 1973; Kazama et al. 1975). However, 206 Mt EDB a G b EDB Mt N Mt c d e f PA g PA i Mt EDB Mt Mt V Mt PA h j Mt k l Fig. 3. Micrographs of the genus Aurantiochytrium spp. a–c Light micrographs showing cells rarely with ectoplasmic net elements (arrows) spread in seawater/pine pollen cultures. d–f Light micrographs of the ovoid-shaped zoospore. g–l Transmission electron micrographs of the electron-dense body and the para-nuclear body (PA) observed in vegetative cells of each strain. (a, d Aurantiochytrium limacinum NIBC SR21; b, e, g, j, l Aurantiochytrium sp. SEK 209; c, f, h, i, k Aurantiochytrium sp. SEK 217). EDB, electron-dense body; G, Golgi body; Mt, mitochondria; N, nucleus; V, vacuole. Bars a–c 30 µm; d–f 5 µm; g–k 0.2 µm; l 0.5 µm Table 3. Profi les of carotenoid pigments of Aurantiochytrium, Oblongichytrium, and Schizochytrium sensu stricto strains Strains Genus Aurantiochytrium Aurantiochytrium limacinum NIBH SR21 Aurantiochytrium sp. SEK217 Aurantiochytrium sp. SEK218 Aurantiochytrium sp. SEK209 Aurantiochytrium sp. N1–27 Aurantiochytrium sp. KH105 Genus Schizochytrium Schizochytrium aggregatum ATCC 28209 Schizochytrium sp. SEK210 Schizochytrium sp. SEK345 Schizochytrium sp. SEK346 Genus Oblongichytrium Oblongichytrium sp. SEK347 Other strains Japonochytrium sp. ATCC 28207 Thraustochytrium striatum ATCC 24473 Astaxanthin Phoenicoxanthin Canthaxanthin Echinenone β-Carotene + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + − − − − − − − − − − − − − − − − + + + + − − + + + + + − + − + − + + + several taxonomic problems for this genus and species have been pointed out. Booth and Miller (1969) reported that the strains had several features different from the original description, but they concluded that both strains should be identified as the same species, that is, Schizochytrium aggre- gatum, and emended the definition of the genus Schizochytrium sensu lato. Alderman et al. (1974) noted that S. aggregatum “seems most probably to be a complex of related organisms which will require considerable effort to separate.” Honda et al. (1999) clearly showed that the 207 K a EDB b G Mt c e d N Mt Mt f Mt Mt g h V i Mt j k l m Fig. 4. Micrographs of the genus Schizochytrium sensu stricto. a–c Light micrographs showing a large cluster of cells with well-developed ectoplasmic nets (arrows) in seawater/pine pollen cultures. d–f Light micrographs of the ovoid-shaped zoospore. g–m Transmission electron micrographs of the electron-dense body and the para-nuclear body (arrowheads) observed in vegetative cells of each strain. (a, d, g, j, m Schizochytrium aggregatum ATCC 28209; b, e, h, k Schizochytrium sp. SEK 210; c, f, i, l Schizochytrium sp. SEK 346). EDB, electron-dense body; G, Golgi body; K, kinetosome; Mt, mitochondria; N, nucleus; V, vacuole. Bars a–c 30 µm; d–f 5 µm; g–l 0.2 µm; m 0.5 µm strains identified as species of Schizochytrium sensu lato appeared at different lineages in the 18S rRNA gene phylogenetic tree; therefore, this molecular phylogeny strongly suggested that the currently applied taxonomic identification of the genus Schizochytrium sensu lato might require reconsideration. The first report of the polyphyly of Schizochytrium sensu lato already showed that members of this genus appeared in three distinct lineages (Honda et al. 1999). However, each of the lineages was composed of only a single strain, so that it was difficult to judge the taxonomic treatment because of the small data set. In the present study, the molecular phylogenetic trees show that each of the lineages is composed of 5 to 11 strains with reasonable genetic variation, which is indicated by the genetic distances (i.e., branch lengths) among the strains in each group. The well-supported monophyletic genera Aplanochytrium and Labyrinthula are composed of 6 and 4 strains in our trees whose genetic variation corresponds to those of three phylogenetic groups of Schizochytrium sensu lato. It is reasonable to treat each phylogenetic group not as the lower (i.e., species) or higher (i.e., family) taxonomic rank but as genus rank. Moreover, we revealed that these three groups could clearly be distinguished by combining phenotypic characteristics obtained by light and electron microscopy, and in profiles of the PUFAs and carotenoid pigments, although it is impossible to distinguish each group using only a single feature. Therefore, these three groups were considered to be three different genera; that is, the genus Schizochytrium sensu stricto, after emendation of the definition, and the two new genera, Aurantiochytrium and Oblongichytrium. The new genus Aurantiochytrium is erected for Aurantiochytrium limacinum, A. mangrovei, and nine strains of unidentified species. The characteristic morphological features of this genus are that the cells in the growth phase tend not to form large colonies regardless of media and not to develop ectoplasmic net elements, and these features can be recognized as a critical difference from Schizochytrium sensu stricto and Oblongichytrium. Under cultivation with liquid nutrient media, small colonies of Aurantiochytrium look like fine grains on the bottom of the flask, whereas the two other genera form large colonies that develop into small balls in the media. The PUFA composition shows that an arachidonic acid level less than 5% was not seen in the other two genera (see Fig. 6). Moreover, the accumulation of astaxanthin is a unique feature among the three groups. 208 Aurantiochytrium A. limacinum* A. sp. KH105* A. sp. N1-27* A. sp. SEK218 a b A. sp. SEK217 c A. sp. SEK209 Schizochytrium S. aggregatum* S. sp. KK17-3* V Mt S. sp. N4-103* S. sp. SEK210 Mt G S. sp. SEK345 S. sp. SEK346 Mt Oblongichytrium PA O. sp. SEK347 Other strains N Japonochytrium sp. U. visurgensis T. aureum Mt V T. striatum 0 (%) 20 40 60 80 100 Mt 20: 4 (n-6) 20: 5 (n-3) 22: 5 (n-6) 22: 5 (n-3) 22: 6 (n-3) AA EPA DPA DPA DHA d Fig. 5. Micrographs of Oblongichytrium sp. SEK 347. a Light micrograph shows a large cluster of cells with well-developed ectoplasmic nets in seawater/pine pollen culture. b Light micrograph of narrow elliptical zoospore. c Transmission electron micrograph of the electrondense body. d Thin section of a young vegetative cell with para-nuclear body. G, Golgi body; Mt, mitochondria; N, nucleus; PA, para-nuclear body; V, vacuole. Bars a 30 µm; b 5 µm; c 0.2 µm; d 0.5 µm Aurantiochytrium limacinum and A. mangrovei share the characteristic feature of releasing amoeboid cells in nutrient media. However, our original strains (i.e., Aurantiochytrium sp. SEK-217, -218, and -209) never released amoeboid cells (data not shown), so this feature is not a distinguishing characteristic for this genus. It is noted that A. limacinum NIBC SR21 is the ex-type strain, but A. mangrovei RCC893 is not, although it was collected from the mangrove area in Hong Kong (K.M. Tsui, personal communication). Unfortunately, the ex-type strain of A. mangrovei has been lost (S. Raghukumar, personal communication), so the phylogenetic position of this species will be revealed after obtaining data from the correctly identified strains collected from the type locality, Goa, in India. The second new genus, Oblongichytrium, is composed of O. minutum, O. multirudimentale, and Oblongichytrium sp. SEK-347, 7-5, and 8-7. This genus is well characterized by Fig. 6. Profiles of polyunsaturated fatty acids of thraustochytrid strains. Bold characters indicate the original isolates whose profiles were analyzed in this study. The genus Aurantiochytrium strains have a content of less than 5% of arachidonic acid and about 80% docosahexaenoic acid; the genus Schizochytrium sensu stricto strains have a content of about 20% arachidonic acids; and the genus Oblongichytrium sp. SEK 347 has a content of about 20% n-3 docosapentaenoic acid. Profiles of other strains are shown: Japonochytrium sp. ATCC 28207, Ulkenia visurgensis ATCC 28208, Thraustochytrium aureum ATCC 34304, Thraustochytrium striatum ATCC 24473. The data of the strains indicated by asterisks were reported by Huang et al. (2003) the narrow ellipsoidal zoospore, ca. 20% docosapentaenoic acid in total PUFA, and accumulation of canthaxanthin and β-carotene, but no astaxanthin. Unfortunately, only Oblongichytrium sp. SEK-347 was examined for PUFA profile and carotenoid pigment in the present study, because the ex-type strain or any other living culture of O. minutum and O. multirudimentale were probably lost and not available from the culture collection. However, the original description of both species clearly showed the characteristic shape of the zoospores (fig. 4g–i in Gaertner 1981; figs. 12–13 in Goldstein 1963). We assume that members of this genus have similar profiles of PUFA and carotenoid pigments. In addition, O. multirudimentale was originally described as a species of the genus Thraustochytrium whose characteristic features in the diagnosis are as follows: (1) 2 to 4 proliferous bodies (= rudiments) of the zoosporangium, and (2) “sub-fusiform or fusiform” ellipsoidal zoospores 209 (Goldstein 1963). It is noteworthy that the division of older sporangia on seawater agar medium was clearly shown (Goldstein 1963). This phenomenon may suggest this species possesses the ability of vegetative cell division. In fact, the dividing “sporangia” look like vegetative cells before zoospore formation in the original micrograph (fig. 18 in Goldstein 1963), because small cells (= immature zoospores) were not observed in the “sporangia.” Therefore, this species should actually be classified as a species of the genus Oblongichytrium, but it is necessary to reexamine the foregoing observation carefully. The members of the third group should be classified in the genus Schizochytrium sensu stricto. This group includes the strain recorded as ATCC 28209 that is identified as S. aggregatum, but it should be noted that this strain is not the ex-type strain. Although the ex-type strain of S. aggregatum has been lost, ATCC 28209 has been distributed from ATCC to a number of researchers and used for investigation with several approaches (Raghukumar 1988b; Honda et al. 1999; Huang et al. 2003). In our observations, the morphology of ATCC 28209 fundamentally agrees with the original description (data not shown), so this strain can be treated as the standard strain of S. aggregatum. Schizochytrium sensu stricto was newly defined with the emendation by adding the three following characteristics: (1) accumulation of ca. 20% arachidonic acid in total PUFA, (2) accumulation of β-carotene without astaxanthin and canthaxanthin, and (3) 18S rRNA gene sequences distinct. Leander and Porter (2001) used their isolate (T91-7) of S. aggregatum, which was located in the clade of the Oblongichytrium and formed a sister-group with O. minutum in the 18S rRNA phylogenetic tree (data not shown). Although the type of S. aggregatum was isolated from a seawater sample in New Haven, Connecticut, USA, strain T91-7 was isolated from the red alga Polysiphonia Grev. in Meridian, Georgia, USA, and strain ATCC 28209 was isolated from seawater in Germany. The correct identification and phylogenetic placement are clearly in need of further investigation for these strains and future isolates from the type locality. These strains are strongly expected to be examined on the morphology of the zoospore and analysis of the PUFA and carotenoid profiles. As already mentioned, taxonomic positions were determined for four species except Schizochytrium octosporum in the genus Schizochytrium sensu lato. Presently there are no molecular phylogenetic data, PUFA, or carotenoid profiles of S. octosporum, but detailed morphological observation was reported in the original description (Raghukumar 1988b). This organism formed large colonies with developed ectoplasmic net elements and released relatively narrow-shaped zoospores (figs. 8 and 13 in Raghukumar 1988b). Hence, we judged that this organism should be classified in the genus Oblongichytrium following the aforementioned taxonomic criteria. It is, however, necessary to reexamine the isolates from the type locality, Rosfjord in Norway. The species of the genera Thraustochytrium and Ulkenia appeared in five and two lineages, respectively, in our phylogenetic tree, and unidentified organisms also formed inde- pendent lineages (see Fig. 2). This observation probably means that further taxonomic rearrangement might be necessary for these lineages. Unfortunately, there are few morphological features reflecting the phylogenetic relationships and not influenced by culture conditions in thraustochytrids. However, the present study strongly suggests that the genus-level clades could be clearly distinguished by combining morphological and chemotaxonomic features, although it is hard to distinguish between groups on the basis of a single feature. Similar situations have occurred in the taxonomy of yeasts and related organisms, and their taxonomic systems have been based on molecular phylogeny (Kregervan Rij 1984; Kurtzman and Fell 1998). Careful analyses and comparisons of both phenotypic and molecular characteristics will establish a well-accepted taxonomic system for the Labyrinthulomycetes. Acknowledgments We are grateful to Dr. T. Yokochi (National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan) for technical advice and kindly providing the data of the PUFA composition of Japonochytrium sp. ATCC 28209, Thraustochytrium aureum ATCC 34304, Thraustochytrium striatum ATCC 24473, and Ulkenia visurgensis ATCC 28208. We thank Drs. T. Nakahara and Y. Kumon (National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan) for technical advice and support concerning analysis by gas chromatography. We also express our thanks to Drs. Y. Yamaoka and M.L. Carmona (National Institute of Advanced Industrial Science and Technology, Hiroshima, Japan) for technical support in analysis by HPLC. We thank Dr. Y. Takao (National Research Institute of Fisheries and Environment of Inland Sea, Hiroshima, Japan) who kindly provided the thraustochytrids strain, SEK 209. We also thank Prof. T. Takaso (University of the Ryukyus, Okinawa, Japan), who kindly supported us in collecting samples in Iriomote Island. 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