Linda A. Amaral Zettler

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.

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.

From Genes to Genomes: Beyond Biodiversity in Spain’s Rio Tinto

Spain’s Rio Tinto, or Red River, an example of an extremely acidic (pH 1.7–2.5) environment with a high metal content, teems with prokaryotic and eukaryotic microbial life. Our recent studies based on small-subunit rRNA genes reveal an unexpectedly high eukaryotic phylogenetic diversity in the river when compared to the relatively low prokaryotic diversity. Protists can therefore thrive in and dominate extremely acidic, heavy-metal-laden environments. Further, because we have discovered protistan acidophiles closely related to neutrophiles, we can hypothesize that the transition from neutral to acidic environments occurs rapidly over geological time scales. How have these organisms adapted to such environments? We are currently exploring the alterations in physiological mechanisms that might allow for growth of eukaryotic microbes at acid extremes. To this end, we are isolating phylogenetically diverse protists in order to characterize and compare ion-transporting ATPases from cultured acidophiles with those from neutrophilic counterparts. We predict that special properties of these ion transporters allow protists to survive in the Rio Tinto.

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.

Towards a molecular phylogeny of colonial spumellarian radiolaria

Gene sequence data from the small-subunit ribosomal RNA coding region were used to explore evolutionary relationships among the colonial spumellarian radiolaria (Polycystinea). Representatives from the two spumellarian families known to form colonies were considered including the following taxa: Sphaerozoidae: Collozoum pelagicum; Collozoum serpentinum; Rhaphidozoum acuferum ; Sphaerozoum punctatum ; and Collosphaeridae: Collosphaera globularis‐huxleyi ; Acrosphaera (circumtexta?); and Siphonosphaera cyathina. Our molecular analyses support the monophyly of the Collosphaeridae in all analyses used, but only distance analyses support the monophyly of the Sphaerozoidae. Within the Sphaerozoidae, two species of Collozoum (C. serpentinum and C. pelagicum) failed to branch together, indicating a more distant relationship than first suggested, a conclusion further supported by recent ultrastructural studies (see adjoining paper). Branching patterns within the Collosphaeridae indicate that Siphonosphaera diverged prior to the split between Collosphaera and Acrosphaera, a result which challenges evidence based on data from the radiolarian fossil record.

Monopylocystis visvesvarai n. gen., n. sp. and Sawyeria marylandensis n. gen., n. sp.: two new amitochondrial heterolobosean amoebae from anoxic environments.

Two new species of heterolobosean amoebae from anoxic environments, Monopylocystis visvesvarai and Sawyeria marylandensis, are described on the basis of light microscopy, electron microscopy, and their phylogenetic affiliation based on analyses of nuclear small-subunit ribosomal RNA gene sequences. Both species lack mitochondria but have organelles provisionally interpreted as hydrogenosomes, and neither can tolerate aerobic conditions. As their conditions of culture do not exclude all oxygen, they may be microaerophiles rather than strict anaerobes. Both species have unusual nucleolar morphologies. Monopylocystis visvesvarai, from a marine sediment, has nucleolar material distributed around the nuclear periphery. It is the first non-aerobic heterolobosean protist for which a cyst is known; the cyst is unmineralized and unornamented except for a single, raised, plugged pore. Sawyeria marylandensis, from an iron-rich freshwater stream, has nucleolar material distributed in one or two parietal masses, which persist during mitosis. In phylogenetic analyses of small-subunit rRNA gene sequences, Monopylocystis visvesvarai, Sawyeria marylandensis and Psalteriomonas lanterna converge to form a single clade of non-aerobic (anaerobic/microaerophilic) heteroloboseans.

Insights on the diversity within a "species" of Thalassicolla (Spumellarida) based on 16S-like ribosomal RNA gene sequencing.

We compared 16S‐like ribosomal RNA (rRNA) coding regions of samples of the solitary spumellarian radiolarian Thalassicolla nucleata collected from the Sargasso Sea and the Pacific Ocean. Sequences derived from these locations showed variability in both length and base‐pair composition. This level of sequence variability is similar to the degree of variability reported in the literature for species‐ or even genus‐level distinctions. Explanations for our results include multiple alleles for the rRNA gene, or the existence of multiple species of Thalassicolla that are morphologically indistinguishable. The seven existing descriptions of Thalassicolla species, including T. nucleata, are discussed in view of these molecular findings and with reference to our current understanding of the physiology and life cycle of the spumellarian radiolaria.

New insights into the phylogeny of the Acantharea based on SSU rRNA gene sequencing

Summary The phylogeny of the Acantharea was examined using small-subunit ribosomal RNA (SSU rRNA) gene sequence analysis of representatives from the Symphyacanthida, Chaunacanthida and the Arthracanthida. Our previous studies showed that Acantharea form a monophyletic group branching as an independent protist lineage among crown groups but not directly related to any of them. The results from this more in-depth molecular analysis of the branching patterns within the Acantharea revealed a phylogeny which is not entirely consistent with morphologybased phylogenies. In particular, the phylogenetic placement of Haliommatidium sp. was in disagreement with its current placement among the Symphyacanthida based on morphological features. Haliommatidium sp. clustered with members of the order Arthracanthida in molecular analyses described herein. In retrospect, Haliommatidium sp. shares several morphological features with the Arthracanthida which support these molecular results.

Fine structure of the colonial radiolarian Collozoum serpentinum (Polycystinea: Spumellaria) with a reconsideration of its taxonomic status and re-establishment of the genus Collophidium (Haeckel)

Abstract Fine structural evidence shows that Collozoum serpentinum differs substantially from two previously examined species Collozoum inerme and Collozoum caudatum . The latter two have spheroidal to prolate central capsules with rounded nuclei and radially arranged lobes of cytoplasm radiating from the center of the central capsules. C. serpentinum has contorted, elongated (greater than 1 mm) cylindrical central capsules, and the intracapsular cytoplasm is highly vacuolated and suspended in a network of denser cytoplasm containing major intracellular organelles including nuclei with irregular shape, mitochondria, and Golgi bodies. This network organization of the intracapsular cytoplasm is distinctly different from the fine structure of Collozoum species examined thus far. These data, combined with molecular genetic evidence (Amaral Zettler et al., 1999), indicate that C. serpentinum is not as closely related to other Collozoum species as was previously thought. Based on this evidence, we suggest that Haeckels original designation of a separate genus ( Collophidium ) is more appropriate for colonial radiolaria with contorted, cylindrical central capsules as represented by C. serpentinum .

A microbial observatory of caterpillars: isolation and molecular characterization of protists associated with the saturniid moth caterpillar Rothschildia lebeau.

LINDA A. AMARAL ZETTLER, ABBY D. LAATSCH, ERIK ZETTLER, THOMAS A. NERAD, JEFFREY COLE, FELIPE CHAVARRIA DIAZ, JOEL DIAZ, DANIEL H. JANZEN, ANA SITTENFELD, OLIVIA MASON and ANNA-LOUISE REYSENBACH The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts 02543, USA, and Sea Education Association, P.O. Box 6, Woods Hole, Massachusetts 02543, USA, and Centro de Biologı́a Molecular, UAM-CSIC, Cantoblanco, Madrid, Spain, and George Mason University, Manassas, Virginia 20110, USA, and American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, USA, and Area de Conservacı́on Guanacaste, Apdo. 169, Liberia 5000, Costa Rica, and Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA, and Centro de Investigacı́on en Biologı́a Celular y Molecular (CIBCM) and Faculty of Microbiology, Universidad de Costa Rica, San José, Costa Rica, and College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, Oregon 97331, USA, and Department of Biology, Portland State University, Portland, Oregon 97201, USA