Øjvind Moestrup

The New Higher Level Classification of Eukaryotes with Emphasis on the Taxonomy of Protists

Abstract. This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional “kingdoms.” The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.

A STUDY OF THE PSEUDO‐NITZSCHIA PSEUDODELICATISSIMA/CUSPIDATA COMPLEX (BACILLARIOPHYCEAE): WHAT IS P. PSEUDODELICATISSIMA? 1

Based on morphological variation found in specimens ascribed to Pseudo‐nitzschia pseudodelicatissima and uncertainty regarding the delineation of P. pseudodelicatissima and P. cuspidata, cultures and field material of diatoms in the P. pseudodelicatissima/cuspidata complex were studied in morphological detail. Four different species were identified. The descriptions of the species P. pseudodelicatissima and P. cuspidata were emended on the basis of studies of type material. In addition, P. calliantha sp. nov. and P. caciantha sp. nov. were described as new species based on morphological and molecular data. The morphological differences among the species were found in characters such as width and shape of the valve, density of fibulae and striae, structural pattern of the poroid hymen, and structure of the girdle bands. The morphological studies were supported by phylogenetic analyses of the nuclear‐encoded internal transcribed spacer 1, 5.8S, and internal transcribed spacer 2 rDNA of 24 strains representing 16 different Pseudo‐nitzschia species. The description of the four species helps to explain the variation observed in mating experiments on cultures originally designated as P. pseudodelicatissima. At least two previous reports of toxin production in species identified as P. pseudodelicatissima have been identified as being caused by P. calliantha, and one additional report of toxin production has been identified as either P. pseudodelicatissima or P. cuspidata.

INTER- AND INTRASPECIFIC VARIATION OF THE PSEUDO-NITZSCHIA DELICATISSIMA COMPLEX (BACILLARIOPHYCEAE) ILLUSTRATED BY RRNA PROBES, MORPHOLOGICAL DATA AND PHYLOGENETIC ANALYSES1

A study of 25 cultures tentatively identified as Pseudo‐nitzschia delicatissima (Cleve) Heiden, and originating from geographically widely distributed locations, showed both morphological and genetic variation among strains. Use of rRNA‐targeted DNA probes on 17 different strains showed large variation in the hybridization patterns. Detailed morphological studies placed the isolates into three groups. The sample on which the neotype of P. delicatissima is based was also examined, and used to establish the morphological identity of P. delicatissima. Phylogenetic analyses of 16 strains, based on sequences of internal transcriber spacer 1 (ITS1), 5.8S and ITS2 of the nuclear‐encoded rDNA, supported the morphological observations and the hybridization studies, and revealed large genetic variation among strains. A combination of the morphological and molecular findings resulted in the description of two new species, P. decipiens sp. nov. and P. dolorosa sp. nov. P. dolorosa has a mixture of one or two rows of poroids in the striae whereas P. delicatissima always has two rows. In addition, P. dolorosa has wider valves and a lower density of poroids. P. decipiens differs from P. delicatissima by a higher density of striae on the valve face as well as a higher density of poroids on the girdle bands. Among the strains referred to P. delicatissima, an epitype was selected. Large genetic variation was found among the P. delicatissima strains and a subdivision into two major clades represent cryptic species.

Diversity, Nomenclature, and Taxonomy of Protists

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Phylogeny of the Bacillariaceae with emphasis on the genus Pseudo-nitzschia (Bacillariophyceae) based on partial LSU rDNA

In order to elucidate the phylogeny and evolutionary history of the Bacillariaceae we conducted a phylogenetic analysis of 42 species (sequences were determined from more than two strains of many of the Pseudo-nitzschia species) based on the first 872 base pairs of nuclear-encoded large subunit (LSU) rDNA, which include some of the most variable domains. Four araphid genera were used as the outgroup in maximum likelihood, parsimony and distance analyses. The phylogenetic inferences revealed the Bacillariaceae as monophyletic (bootstrap support ≥90%). A clade comprising Pseudo-nitzschia, Fragilariopsis and Nitzschia americana (clade A) was supported by high bootstrap values (≥94%) and agreed with the morphological features revealed by electron microscopy. Data for 29 taxa indicate a subdivision of clade A, one clade comprising Pseudo-nitzschia species, a second clade consisting of Pseudo-nitzschia species and Nitzschia americana, and a third clade comprising Fragilariopsis species. Pseudo-nitzschia as presently defined is paraphyletic and emendation of the genus is probably needed. The analyses suggested that Nitzschia is not monophyletic, as expected from the great morphological diversity within the genus. A cluster characterized by possession of detailed ornamentation on the frustule is indicated. Eighteen taxa (16 within the Bacillariaceae) were tested for production of domoic acid, a neurotoxic amino acid. Only P. australis, P. multiseries and P. seriata produced domoic acid, and these clustered together in all analyses. Since Nitzschia navis-varingica also produces domoic acid, but is distantly related to the cluster comprising the Pseudo-nitzschia domoic acid producers, it is most parsimonious to suggest that the ability of species in the Bacillariaceae to produce domoic acid has evolved at least twice.

Parvilucifera infectans norén et moestrup gen. et sp. nov. (perkinsozoa phylum nov.): a parasitic flagellate capable of killing toxic microalgae

Summary The toxic dinoflagellate Dinophysis, collected on the Swedish West Coast, was found to contain round bodies previously interpreted as the result of sexual reproduction. After two weeks of darkness in the refrigerator, all Dinophysis had died, however, and round bodies were present. These proved to be sporangia of a parasitic protist, here named Parvilucifera infectans gen. et sp. nov. Its identity was examined by LM, EM, and DNA sequencing. It is related to Perkinsus, an oyster-killing protist, and Colpodella, a phagocytic protist. Perkinsus has been indicated by 18S rRNA sequencing to be related to dinoflagellates, and the opportunity was taken to examine the ultrastructure of the flagellar apparatus of Parvilucifera in detail. Parvilucifera and its allies, known as perkinsids, share features with both dinoflagellates and apicomplexans. They do not fit readily into any of these groups but appear to form a missing link between them. They are described as a taxon on level with the other alvelolate phyla, as Perkinsozoa phylum nov. Infection studies showed that Parvilucifera infectans infects several other dinoflagellates, notably Alexandrium spp. which are responsible for PSP (paralytic shellfish poisoning). A discussion of the ecological role, in terms of biocontrol of harmful algal blooms, is included.

ON THE IDENTITY OF KARLODINIUM VENEFICUM AND DESCRIPTION OF KARLODINIUM ARMIGER SP. NOV. (DINOPHYCEAE), BASED ON LIGHT AND ELECTRON MICROSCOPY, NUCLEAR-ENCODED LSU RDNA, AND PIGMENT COMPOSITION1

An undescribed species of the dinoflagellate genus Karlodinium J. Larsen (viz. K. armiger sp. nov.) is described from Alfacs Bay (Spain), using light and electron microscopy, pigment composition, and partial large subunit (LSU) rDNA sequence. The new species differs from the type species of Karlodinium (K. micrum (Leadbeater et Dodge) J. Larsen) by lacking rows of amphiesmal plugs, a feature presently considered to be a characteristic of Karlodinium. In K. armiger, an outer membrane is underlain by a complex system of cisternae and vacuoles. The pigment profile of K. armiger revealed the presence of chlorophylls a and c, with fucoxanthin as the major carotenoid. Phylogenetic analysis confirmed K. armiger to be related to other species of Karlodinium; thus forming a monophyletic genus, which, in the LSU tree, occupies a sister group position to Takayama de Salas, Bolch, Botes et Hallegraeff. The culture used by Ballantine to describe Gymnodinium veneficum Ballantine (Plymouth 103) was examined by light and electron microscopy and by partial LSU rDNA. Ultrastructurally, it proved identical to K. micrum (cultures Plymouth 207 and K. Tangen KT‐77D, the latter also known as K‐0522), and in LSU sequence, differed in only 0.3% of 1438 bp. We consider the two taxa to belong to the same species. This necessitates a change of name for the most widely found species, K. micrum, to K. veneficum. The three genera Karlodinium, Takayama, and Karenia constitute a separate evolutionary lineage, for which the new family Kareniaceae fam. nov. is suggested.

Detection of an anatoxin-a(s)-like anticholinesterase in natural blooms and cultures of cyanobacteria/blue-green algae from Danish lakes and in the stomach contents of poisoned birds.

Ten natural bloom samples of cyanobacteria from the Danish lakes Knud sø (5), Ravn sø (4), and Salten Langsø (1) collected during 1993-1995 were assayed for toxicity by mouse bioassay, for acetylcholinesterase inhibiting activity by a colorimetric method, and for microcystins by enzyme-linked immunosorbent assay. In the mouse bioassay, seven samples were neurotoxic, two were non-toxic and one gave a protracted toxic response. One of the non-toxic and the single protracted toxic sample both contained anticholinesterase activity equivalent to 4 micrograms anatoxin-a(s) g-1. The neurotoxic samples contained equivalents to 20-3300 micrograms anatoxin-a(s) g-1. The highest anticholinesterase activities (equivalent to 2300 and 3300 micrograms anatoxin-a(s) g-1, respectively) were found in samples collected from Lake Knud sø in connection with bird-kills in 1993 and 1994. Small amounts of microcystins (0.1-0.9 microgram g-1) were detected in all samples but one. All Lake Knud sø and Lake Ravn sø samples were dominated by Anabaena lemmermannii, and the Lake Salten Langsø sample by several species of Anabaena. Gel filtration profiles indicated similarity between the toxic component from the Lake Knud sø 1994 bloom with registered bird-kills and anatoxin-a(s) isolated from Anabaena flos-aquae NRC-525-17. Anticholinesterase-producing cultures of A. lemmermannii were isolated from the Lake Knud sø 1993 bloom. These laboratory cultures produced anatoxin-a(s) equivalents of 29-743 micrograms g-1. Other cultures of A. lemmermannii isolated from Lake Knud sø and Lake Ravn sø were hepatotoxic or non-toxic. Dead birds collected from Lake Knud sø during the neurotoxic 1993 Anabaena bloom possibly died from cyanobacterial toxicosis. The stomach contents contained colonies and single trichomes of Anabaena, and anticholinesterase activities equivalent to 2.1-89.7 micrograms anatoxin-a(s) kg-1 body weight and microcystins (53-95 ng kg-1) were also detected.

THE MARINE DINOFLAGELLATE ALEXANDRIUM OSTENFELDII: PARALYTIC SHELLFISH TOXIN CONCENTRATION, COMPOSITION, AND TOXICITY TO A TINTINNID CILIATE1

The composition of paralytic shellfish toxins in the marine dinoflagellate Alexandrium ostenfeldii (Paulsen) Balech et Tangen grown in unialgal culture was determined by high‐performance liquid chromatography. The toxin profile revealed that the low‐potency sulfamate toxin B2 was dominant (90 molar % of total toxins), but small amounts of the weakly toxic 21‐N‐sulfocarbamoyl derivatives C1+2 and trace amounts of the carbamate toxins GTX2 and GTX3 were also present. The mammalian toxicity was confirmed by a modification of the conventional AOAC mouse bioassay (0.6–1.4 pg STXeq· cell‐1). The acute toxicity to a potential predator, the tintinnid ciliate Favella ehrenbergi (Clap, et Lach.) Jörg., was also investigated. The ciliate was able to graze on A. ostenfeldii when the cell concentration of the dinoflagellate was low (<2000 cells · mL‐1). At higher concentrations the ciliate was affected by exudates (presumably PSP toxins) that induced backward swimming followed by swelling and lysis of the cell. Fluorescence microscopy of calcofluor‐stained cells was employed as an easy and rapid method to identify this and other thecate dinoflagellates.

An ultrastructural study of the flagellatePyramimonas orientalis with particular emphasis on Golgi apparatus activity and the flagellar apparatus

SummaryThe prasinophycean flagellatePyramimonas orientalis has been examined by light and electron microscopy of wild and cultured material. The many different scales which cover all cell surfaces, including the flagella, are described; their synthesis and assembly in the two Golgi bodies have been examined. The Golgi bodies work simultaneously to produce all-at least five—scale categories, including hollow hair shaped scales. From the Golgi system the scales become transported to a special container—a reservoir—in which they, in an unknown way, separate and become arranged in the same pattern as on the body surface. From the reservoir, the scales move through a duct to the cell surface, apparently together with the subtending membrane, which thus becomes incorporated in the plasmalemma or the flagellar membrane. The liberation process, which differs from that of other species ofPyramimonas examined, is illustrated diagrammatically, starting at two extensions of ER from the nuclear envelope.The flagellar apparatus possesses a flagellar root system of the green algal type, a finding of phylogenetic significance. Furthermore, near the flagellar transition region a structure was observed, which at present is known from certain “brown” groups of algae, but never from any green flagellate. The taxonomic implications are discussed briefly, and a virus attacking the nuclear area of the cell is reported. Very surprisingly two different sizes of the virus were found, which may be different stages of the same “organism”.