Mark A. Farmer

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.

Diversity, Nomenclature, and Taxonomy of Protists

the origin of echolocation and flight in bats. Nature 403:188– 192. van Rheede, T., T. Bastiaans, D. N. Boone, S. B. Hedges, W. W. de Jong, and O. Madsen. 2006. The platypus in its place: nuclear genes and indels confirm the sister group relation of monotremes and therians. Mol. Biol. Evol. 23:587–597. Waddell, P. J., H. Kishino, and R. Ota. 2001. A phylogenetic foundation for comparative mammalian genomics. Genome Informatics 12:141– 154. Waddell, P. J., N. Okada, and M. Hasegawa. 1999. Towards resolving the interordinal relationships of placental mammals. Syst. Biol. 48:1–5. Waddell, P. J., and S. Shelley. 2003. Evaluating placental inter-ordinal phylogenies with novel sequences including RAG1, γ -fibrinogen, ND6, and mt-tRNA, plus MCMC-driven nucleotide, amino acid, and codon models. Mol. Phylogenet. Evol. 28:197–224.

Character evolution in heterotrophic euglenids

This article attempts to describe the key morphological innovations associated with the evolutionary transitions between bacteriotrophy, eukaryotrophy, phototrophy, and osmotrophy in euglenids. Attention was focused on heterotrophic euglenids in an effort to establish a robust phylogenetic hypothesis for the group as a whole. We present a cladistic analysis of a large morphological data set from the following taxa: Petalomonas, Entosiphon, Lentomonas, Ploeotia, Dinema, Distigma, Rhabdomonas, Menoidium, Peranema, Urceolus, Eutreptia, and Euglena. The majority of the 37 characters and 97 states recognized were associated with the pellicle, the feeding apparatus, and the flagellar apparatus. In addition to having pellicle strips, Petalomonas cantuscygni possessed mitochondrial inclusions that were strikingly similar to the kinetoplasts found in kinetoplastids. Dinema sulcatum held a pivotal position in the phylogenetic tree and possessed many characters that bridged bacteriotrophic taxa with eukaryotrophic taxa. Distigmids and rhabdomonads formed a clade of osmotrophs that descended from eukaryotrophic ancestors, while Urceolus cyclostomus possessed a feeding apparatus, a putative photoreception apparatus and cytoskeletal features that clearly linked the phototrophs to eukaryotrophic ancestors. Evolutionary implications that emerged from these results were discussed.

Comparative Morphology of the Euglenid Pellicle. II. Diversity of Strip Substructure

ABSTRACT. The morphological diversity associated with the strip substructure of the euglenid pellicle was examined, and after identifying characters and states, we outlined hypotheses about their evolution. We have attempted to standardize terms necessary for analytical comparisons of strips by providing a glossary and comparing published synonyms. Most of the substructural diversity found in euglenids is demonstrated with 13 representative taxa. Strips are generally composed of two subcomponents: frames and projections. Frames support the basic shape of strips and many can be described as either S‐shaped, plateau‐shaped, M‐shaped, or A‐shaped. Projections branch laterally from the frames, are usually periodic, and can be described as thread‐like structures, an indented plate, tooth‐like structures, and plate‐like structures. The ancestral state included strips that were few in number, flat, and fused. The strips became S‐shaped and disjoined in the lineage leading to most euglenid taxa. These strips became secondarily flattened and fused in one lineage. In some lineages of phototrophs, the strips became increasingly robust. Two strips of different morphology formed the repeating pellicular unit or doublet in four taxa. These doublets evolved convergently at least three times and may provide insights into developmental patterns of the cytoskeleton.

TRENDS IN THE EVOLUTION OF THE EUGLENID PELLICLE

Abstract Trends in the evolution of the euglenid pellicle were described using phylogenetic methods on 18S rDNA, morphological, and combined data from 25 mostly phototrophic taxa. The tree topology from a total‐evidence analysis formed a template for a synthetic tree that took into account conflicting results derived from the partitioned datasets. Pellicle character states that can only be observed with the assistance of transmission and scanning electron microscopy were phylogenetically mapped onto the synthetic tree to test a set of previously established homology statements (inferences made independently from a cladogram). The results permitted us to more confidently infer the ancestral‐derived polarities of character state transformations and provided a framework for understanding the key cytoskeletal innovations associated with the evolution of phototrophic euglenids. We specifically addressed the character evolution of (1) the maximum number of pellicle strips around the cell periphery; (2) the patterns of terminating strips near the cell posterior end; (3) the substructural morphology of pellicle strips; (4) the morphology of the cell posterior tip; and (5) patterns of pellicle pores on the cell surface.

Comparative morphology of the euglenid pellicle. I. Patterns of strips and pores.

Abstract In anticipation that improved knowledge of euglenid morphology will provide robust apomorphy-based definitions for clades, transmission and scanning electron microscopy were used to reveal novel morphological patterns associated with the euglenid pellicle. In some taxa, the number of pellicle strips around the cell periphery reduces as discrete whorls at the anterior and posterior ends of the cell. The number of whorls at either end varies between selected euglenid taxa but is invariant within a taxon. The pattern of strip reduction associated with these whorls is shown to have at least three evolutionarily linked states: exponential, pseudoexponential, and linear. Two general equations describe these states near the posterior end of euglenid cells. Exponential patterns of strip reduction near the anterior end are described by a third equation. In addition, several euglenid taxa were found to possess conspicuous pellicle pores. These pores are arranged in discrete rows that follow the articulation zones between adjacent strips. The number of strips between rows of pores varies between taxa and displays a series of consecutive character states that differ by a power of two. The patterns of pores may not only have phylogenetical and taxonomical value but may provide morphological markers for following strip maturation during cytoskeletal reproduction.

Ultrastructure of Ditrichomonas honigbergii N. G., N. Sp. (Parabasalia) and its relationship to amitochondrial protists

ABSTRACT. Ditrichomonas honigbergii n. g., n. sp. is a small trichomonad flagellate that has three emergent flagella arising from four basal bodies, a parabasal apparatus (single dictyosome with associated striated flagellar rootlets), a microtubular axostyle, a short undulating membrane, and hydrogenosomes. Cultures of D. honigbergii were isolated from the sediments of a freshwater lake and there is no known metazoan host. Cells form walled cysts with internalized flagella and go through all phases of the life cycle (excystment, binary division, encystment) without any perturbations to the culture medium. Ditrichornonas honigbergii is capable of ingesting and digesting bacteria by phagocytosis. These facts suggest that D. honigbergii may be a free‐living inhabitant of oxygen‐reduced environments. The structure of D. honigbergii is similar to that of retortamonads and the relationship of trichomonads to other amitochondrial protists is discussed.

EVOLUTION OF PHACUS (EUGLENOPHYCEAE) AS INFERRED FROM PELLICLE MORPHOLOGY AND SSU rDNA

This research integrates a large morphological data set into a molecular context. Nineteen pellicle characters and 62 states from 13 euglenid taxa were analyzed cladistically. The pellicle morphology of Euglena tripteris (Klebs), Lepocinclis ovata (Conrad), Phacus brachykentron (Pochmann), P. oscillans (Klebs), P. pyrum (Stein), and P. triqueter (Dujardin) is described comprehensively. These data are compared with new information on the pellicle morphology of Euglena acus (Ehrenberg), E. stellata (Mainx), and Peranema trichophorum (Stein) in addition to published data on Entosiphon sulcatum (Dujardin), Euglena gracilis (Klebs), Distigma proteus (Pringsheim), and Petalomonas cantuscygni (Cann and Pennick). Nuclear small subunit (SSU) rDNA sequences provided an independent test for establishing a robust organismal pedigree of the same taxa. A synthetic tree derived from the combined phylogenetic analyses of pellicle morphology and SSU rDNA enabled us to parsimoniously map morphological character states. This approach demonstrated the utility of pellicle morphology for inferring phylogenetic relationships of euglenids and establishing apomorphy‐based clade definitions. Three robust clades with unambiguous pellicle‐based apomorphies can be recognized within taxa traditionally classified as Phacus: (1) L. ovata and P. pyrum, (2) E. tripteris and P. triqueter, and (3) P. brachykentron and P. oscillans. Taxonomic concerns that emerged from these results are discussed.

Cellular localization of antiviral polyoxometalates in J774 macrophages

The cellular localization of the polyoxometalates, K12H2[P2W12O48].24H20 (JM 1591), K10[P2W18-Zn4(H2O)2O68].20H2O (JM 1596), and [Me3NH]8[Si2W18Nb6O77] (JM 2820) were examined in cultured J774 cells by inhibition of cellular uptake of acetylated low-density lipoprotein (LDL) and by electron microscopy. All three polyoxometalates inhibited the cellular uptake of acetylated LDL, suggesting that the polyoxometalates block the association of acetylated LDL with cellular scavenger receptors. Fluorescence microscopy showed increased numbers of vacuoles in the presence of polyoxometalates, suggesting their uptake by cells. Using scanning electron microscopy (SEM), no significant cell surface morphological differences were observed between treated and non-treated J774 cells, suggesting that the compounds are not toxic to J774 cells up to a concentration of 200 micrograms/ml. Transmission electron microscopy (TEM) revealed large amounts of high electron dense granules were observed in the ramifying system of tubular cavities and vacuoles. TEM-energy dispersive spectroscopy (EDS) X-ray microanalysis was unable to differentiate the dense particles, most likely because the amount of tungsten in the cells was below the limit of detection. X-ray microanalysis conducted using the SEM-wavelength dispersive spectroscopy (WDS) detected tungsten, averaging 0.45 +/- 0.16% (mean +/- S.D.), in the J774 cells treated with JM 2820, suggesting that this polyoxometalate was taken up by the macrophages or was bound to their surface. Polyoxometalates interact at the cell surface and appear to be taken up by J774 macrophages. The cellular localization of polyoxometalates may be associated with anti-HIV activity.

Three-dimensional confocal microscopy and visualization of the in situ cornea

The in situ cornea is an ideal test specimen to evaluate techniques for 3D reconstruction and visualization of unstained, unfixed, transparent living tissues from a stack of optical sections. The 0.4 mm thick transparent specimen has been optically sectioned into 365 sections using a confocal laser scanning microscope (CLSM) with a water immersion objective. Depth-dependent light attenuation due to absorption and scatter within the specimen was manually compensated at each sampled section. A water immersion microscope minimized the spherical aberrations that would have occurred with the use of an oil immersion objective. Isometric sampling resulted in near-cubic voxels, which compensated for the reduced microscopic resolution in the z axis as compared to x and y resolution.