Thomas A. Nerad

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.

Small subunit ribosomal RNA+ of Hexamita inflata and the quest for the first branch in the eukaryotic tree

A phylogenetic analysis of the small subunit ribosomal RNA (16S-like rRNA) coding region from Hexamita inflata demonstrates that parasitism alone cannot explain early diverging eukaryotic lineages. Parasitic and free-living diplomonads, as well as trichomonads and microsporidia, diverge at the base of the eukaryotic tree. The relative branching order of diplomonads, trichomonads and microsporidia is influenced by outlying prokaryotic taxa with different G+C compositions in their rRNA coding regions. The high G+C prokaryotes position Giardia lamblia at the base of the eukaryotic tree but split diplomonads into a paraphyletic group. When the outlying groups are restricted to rRNAs with nominal G+C compositions, diplomonads form a monophyletic group that diverged after the microsporidia and trichomonads. This unstable branching pattern correlates with unusual nucleotide compositions in the rRNAs of G. lamblia (75% G+C) and Vairimorpha necatrix (35% G+C). In contrast, the 51% G+C composition of the H. inflata rRNA is typical of other eukaryotic rRNAs. Its divergence after trichomonads is strongly supported by bootstrap replicates in distance analyses that do not include G. lamblia. Because of a low G+C composition in its rRNA coding region, the phylogenetic placement of V. necatrix is uncertain and the identity of the deepest branching eukaryotic lineage is ambiguous.

Phylogeny of trichomonads inferred from small-subunit rRNA sequences.

ABSTRACT. Small subunit (16S‐like) ribosomal RNA sequences were obtained from representatives of all four families constituting the order Trichomonadida. Comparative sequence analysis revealed that the Trichomonadida are a monophyletic lineage and a deep branch of the eukaryotic tree. Relative to other early divergent eukaryotic assemblages the branching pattern within the Trichomonadida is very shallow. This pattern suggests the Trichomonadida radiated recently, perhaps in conjunction with their animal hosts. From a morphological perspective the Devescovinidae and Calonymphidae are considered more derived than the Monocercomonadidae and Trichomonadidae. Molecular trees inferred by distance, parsimony and likelihood techniques consistently show the Devescovinidae and Calonymphidae are the earliest diverging lineages within the Trichomonadida, however bootstrap values do not strongly support a particular branching order. In an analysis of all known 16S‐like ribosomal RNA sequences, the Trichomonadida share most recent common ancestry with unidentified protists from the hindgut of the termite Reticulitermes flavipes. The position of two putative free‐living trichomonads in the tree is indicative of derivation from symbionts rather than direct descent from some free‐living ancestral trichomonad.

A molecular reassessment of the Leptomyxid amoebae.

Leptomyxid amoebae encompass a diverse assemblage of amoeboid protists that have been implicated as encephalitis-causing agents. This characteristic is attributed to recent studies identifying new members of the Leptomyxidae, in particular, Balamuthia mandrillaris, that cause the disease. Their morphologies range from limax to plasmodial, as well as reticulated and polyaxial. Although systematic studies have identified B. mandrillaris as a new member of the Leptomyxidae, its precise placement within the leptomyxids is uncertain. To further assess the taxonomic placement of Balamuthia among the leptomyxid amoebae and to determine whether the members of the Leptomyxida form a monophyletic assemblage, we have sequenced 16S-like rRNA genes from representatives of three leptomyxid families. Our phylogenetic analyses revealed that current members of the order Leptomyxida do not constitute a monophyletic assemblage. Our analyses clearly show that Gephyramoeba, as well as Balamuthia do not belong in the order Leptomyxida. We highlight where molecular data give differing insights than taxonomic schemes based on traditional characters.

Characterization of a New Pathogenic Acanthamoeba Species, A. byersi n. sp., Isolated from a Human with Fatal Amoebic Encephalitis

Acanthamoeba spp. are free‐living amoebae that are ubiquitous in natural environments. They can cause cutaneous, nasopharyngeal, and disseminated infection, leading to granulomatous amebic encephalitis (GAE) in immunocompromised individuals. In addition, they can cause amoebic keratitis in contact lens wearers. Acanthamoeba GAE is almost always fatal because of difficulty and delay in diagnosis and lack of optimal antimicrobial therapy. Here, we report the description of an unusual strain isolated from skin and brain of a GAE patient. The amoebae displayed large trophozoites and star‐shaped cysts, characteristics for acanthamoebas belonging to morphology Group 1. However, its unique morphology and growth characteristics differentiated this new strain from other Group 1 species. DNA sequence analysis, secondary structure prediction, and phylogenetic analysis of the 18S rRNA gene confirmed that this new strain belonged to Group 1, but that it was distinct from the other sequence types within that group. Thus, we hereby propose the establishment of a new species, Acanthamoeba byersi n. sp. as well as a new sequence type, T18, for this new strain. To our knowledge, this is the first report of a Group 1 Acanthamoeba that is indisputably pathogenic in humans.

The Nucleariid Amoebae: More Protists at the Animal‐Fungal Boundary

Abstract Nucleariid amoebae are naked amoebae, generally characterized by a spherical or sometimes flattened body with radiating filopodia. Most species preferentially consume algal prey or cyanobacteria. Phylogenetic analyses of the small-subunit rRNA coding regions from four nucleariid amoebae place these species near the origin of the animal-fungal divergence, together with the choanoflagellate-Corallochytrium and the ichthyosporean clades. The species Nuclearia delicatula, N. moebiusi, and N. simplex form a monophyletic group, while ATCC 30864, tentatively but possibly incorrectly assigned to Nuclearia sp., represents a separate line of descent. These nucleariids are unrelated to the lineage containing the testate filose amoebae (Testaceafilosia). Our findings expand the morphological and phylogenetic diversity of protists at the animal-fungal divergence.

Malawimonas jakobiformis n. gen., n. sp. (Malawimonadidae n. fam.): A Jakoba‐like Heterotrophic Nanoflagellate with Discoidal Mitochondrial Cristae

Malawimonas jakobiformis n. gen., n. sp., is established for a bacterivorous heterotrophic nanoflagellate isolated from the Malawi shore of Lake Nyasa (eastern Africa). Trophic stages observed were anteriorly biflagellate and naked. The posterior flagellum of a trophic cell resided in a conspicuous groove on the ventral surface, and bore a prominent vane. A Golgi stack and a mitochondrion with discoidal cristae were present anterior to the nucleus. The kinetid consisted of two short, slightly separated basal bodies, four microtubular roots, and associated fibers and bands. The three microtubular roots associated with the posterior basal body were associated with the ventral groove, while the single root associated with the anterior basal body gave rise to secondary cytoskeletal microtubules. Dividing cells became rounded, with persistent flagella. Cysts were uninucleate, and had thin organic walls without clearly differentiated apertures or ornamentation but with conspicuous attachment pads. Kinetid elements were present within cysts. On the basis of microscopical features, especially those of the kinetid, the nearest relatives of M. jakobiformis are the mitochondriate “jakobid” protists (families Histionidae and Jakobidae) and the amitochondriate retortamonads. Malawimonadidae n. fam. is established to accommodate this species.

Two New Small-Subunit Ribosomal RNA Gene Lineages within the Subclass Gymnamoebia

Abstract Phylogenetic analysis of small-subunit ribosomal RNA gene sequences for gymnamoebae of the families Vexilliferidae, Paramoebidae, and Vannellidae identified two distinct lineages that are supported by gross morphological characters. This analysis indicates that paramoebids and vexilliferids are part of one lineage and that vannellids belong to another. A shared morphological character unique to the paramoebid/vexilliferid lineage members is the presence of dactylopodiate subpseudopodia. However, cell surface structures, normally used for taxonomic discrimination, range from simple hair-like filaments without any apparent organization (Neoparamoeba), to hexagonal glycostyles (Vexillifera) or more elaborate surface scales (Korotnevella). Taxa within the vannellid lineage all lack subpseudopodia and appear flabellate, spatulate or linguiform while in locomotion. Cell surface structures of taxa within the vannellid lineage range from filaments organized into hexagonal arrays (Lingulamoeba, Platyamoeba) to pentagonal glycostyles (Clydonella, Vannella). Vannellid lineage members of the genera Clydonella and Lingulamoeba were studied at the level of electron microscopy. Unique cell surface features validate these as genera distinct from Vannella and Platyamoeba. Genetic and ultrastructural data are used to discuss the phylogenetic interrelationships for the taxa studied.

Spironucleus vortens n. sp. from the freshwater angelfish Pterophyllum scalare: morphology and culture.

ABSTRACT. A new diplomonad flagellate, Spironucleus vortens n. sp., is described from the intestinal lumen of the freshwater angelfish, (Pterophyllum scalare), bred in Florida. Live organisms are pyriform, and measure 12.5–20.5 μm long by 5.0–11.2 üm wide. Scanning electron microscopy shows that the trophozoite bears two compound lateral longitudinal ridges, each originating posterior to three emerging anterior flagella, and continuing posteriorly to the emergence of the posterior flagellum. Each ridge comprises a broad central part, surrounded by a peripheral ridge. At the opening of the flagellar pocket, the broader right peripheral ridge crosses to the other side of the body, and then back again. The posterior end of the body bears two papillae. Transmission electron microscopy shows that the compound lateral ridges are supported by microtubules, and bear microfibrillar structures in discrete longitudinal plaques. The serendipitous growth of S. vortens in a culture system with lip tumor tissue, facilitated axenic cultivation in a modified TYM medium (trypticase, yeast extract, maltose). The flagellate is now routinely maintained in an axenic TYI‐S‐33 medium (trypticase, yeast extract, iron serum), and is stabilized in the cryopreserved state. Spironucleus vortens is an aerotolerant anaerobe that can be cultured at 25° C, 28° C and 30° C.

Reclinomonas americana N. G., N. Sp., a new freshwater heterotrophic flagellate.

ABSTRACT. A new heterotrophic flagellate has been discovered from sites in Maryland, Michigan and Wyoming. The flagellate resides within a lorica constructed of a meshwork of intertwined fibrils with the outer surface invested with nail‐shaped spines. The organism “reclines” within the lorica with its ventral aspect directed upward, and has two heterodynamic flagella, neither of which bears mastigonemes. One flagellum is directed upward and the other is arched over the ventral aspect of the body. Ingestion of bacteria takes place at the left posterior half of the cell. The organism is anchored to the lorica on the right posterior side by a series of regularly spaced cytoplasmic bridges and at the left anterior of the cell by a cytoplasmic appendage similar to the “languette cytoplasmique” found in some bicosoecids. The right side of the cell is raised into a flattened lip with the outer margin reinforced by a ribbon of microtubules. The new flagellate has mitochondria with tubular cristae and lacks a Golgi. A new genus is created to accommodate both the new flagellate described herein and Histiona campanula Penard. A new family is proposed to include the new genus and Histiona.