On the Identity of the Amoeboflagllates Didascalus throntoni and Adelphamoeba galeacystis

ABSTRACT. Didascalus thorntoni , Singh 1952 has been classified alternately as a separate genus or as a species of Naegleria. In the 18th edition of the American Type Culture Collection catalogue it is classified as Naegleria thorntoni. To resolve the question of its identity we have used riboprinting and sequencing of the small subunit ribosomal DNA. The results indicate that D. thorntoni does not belong to the genus Naegleria. The sequence of the small subunit ribosomal DNA differs only in 20 nucleotides (1%) from that of the Paratetramitus jugosus. The difference is much smaller than between some species of Naegleria. Therefore, it is not clear whether D. thorntoni should be considered as a species of Paratetramitus or as a separate genus. The strain used in different laboratories as the type strain of Adelophamoeba gleacystis has been identified as a Naegleria strain. We believe that the type strain of A. galeacystis was mislabeled prior to submission to the American Type Culture Collection and to the Culture Collection of Algae and Protozoa. A recent isolate, which on the basis of morphology was identified as a strain of A. Galeacystis , has the identical small subunit ribosomal DNA sequence as D. throntoni. Our results prove Page was right when he stated that Adelphamoeba might be a synonym of Didascalus.     

The flagellar apparatus of spermatozoa in fish. Ultrastructure and evolution

Chondrichthyes possess an evolved type of spermatozoa. Their flagellar apparatus is characterized by the presence of flagellar roots which form the axis of the midpiece, and the existence of one or two lateral elements associated with the axoneme. Osteichthyes, mainly teleosteans, show a great diversity of spermatic forms. The primitive spermatozoon with a 9 + 2 pattern flagellum is common. The primitive spermatozoon has evolved along different lines. The spermatic diversity which results from this is mainly evident in the structure of the flagellar apparatus. In the animal kingdom the primitive spermatozoon with a 9 + 2 pattern flagellum, present in primitive metazoa, is retained in phyla where external fertilization is maintained. The main evolutionary tendencies--elongation, aflagellarity or biflagellarity--are generally connected with the acquisition of internal fertilization. These evolutionary tendencies are found in teleosteans. It is not possible to link aflagellarity or biflagellarity of the gamete in certain fishes to this method of fertilization. Only the elongation of the spermatozoon is connected, in certain cases, with internal fertilization, but this cannot be taken as general.     

Characterization of Pharyngomonas kirbyi (= "Macropharyngomonas halophila" nomen nudum), a very deep-branching, obligately halophilic heterolobosean flagellate

The tetraflagellate Pharyngomonas is among the most commonly reported morphotypes of halophilic protozoa. We have established two cultures of Pharyngomonas kirbyi, SD1A and AS12B, from 300‰ and 210‰ salinity waters from the USA and Australia, respectively. 18S rRNA gene phylogenies confirm that Pharyngomonas is the same entity as 'Macropharyngomonas' (nomen nudum), and represents the deepest branch in the heterolobosean lineage. Pharyngomonas kirbyi (Strain SD1A) has flattened/discoidal cristae, and lacks conspicuous Golgi dictyosomes. It also has a heterolobosean 'double bikont' flagellar apparatus, with two right roots, each associated with an 'I' fibre and part of a rhizoplast-like complex. One right root splits shortly after its origin, and supplies most of the microtubules that support both the ventral groove, and the sub-anterior cytopharynx. Interestingly, Pharyngomonas has some potentially ancestral features not found in typical Heterolobosea, including elongated left roots associated with multilayered 'C' fibres, orthogonal basal bodies, and a spur structure that might represent a 'B' fibre homolog. Both isolates are obligate halophiles that grow best at 100-200‰ salinity and do not grow below 75‰ salinity. Pharyngomonas is therefore of considerable evolutionary importance, both as a deep-branching, plesiomorphic heterolobosean, and a borderline extreme halophile.     

Ultrastructure of the flagellar apparatus of Oxyrrhis marina

Summary— Oxyrrhis marina, like all dinoflagellates, possesses one transverse and one longitudinal flagellum, which show structural differences. The transverse flagellum contains a small fibre, 20 nm in diameter, associated with doublet no.7, whereas the longitudinal flagellum is substantially by a large (200–300 nm) hollows structure closely resembling the paraflagellar rod described by several authors in kinetoplastidae and in euglenoids. This structure is made up of a hemicylindrical network of filaments which are often linked on one side to the outer doublet no. 4, and on the other side to a dense plate. Another thinner filamentous network closes this hemicyclinder. In cross-section, the wall of this structure is made up of 8 filaments 2–4 nm in diameter that show a thicker periodic structure. In longitudinal section the same filaments appear arranged in periodic rhombus meshes or a helicoidal pattern, depending on the orientation of the section relative to the axoneme.     

Primary Structure of the Hydrogenosomal Malic Enzyme of Trichomonas vaginalis and Its Relationship to Homologous Enzymes1

ABSTRACT. The complete nucleotide sequence has been established for two genes (maeA and maeB) coding for different subunits of the hydrogenosomal malic enzyme [malate dehydrogenase (decarboxylating) EC 1.1.1.39] of Trichomonas vaginalis. Two further genes (maeC and maeD) of this enzyme have been partially sequenced. The complete open reading frames code for polypeptides of 567 amino acids in length. These two open reading frames are similar with less than 12 percent pairwise nucleotide differences and less than 9 percent pairwise amino acid differences. The open reading frames of the two partially sequenced genes correspond to the amino-terminal part of the polypeptides coded and are similar to the corresponding parts of the completely sequenced ones. The deduced translation products of the two complete genes differ in their calculated pI values by 1.5 pH unit. The genes code for polypeptides which contain 12 or 11 amino-terminal amino-acyl residues not present in the proteins isolated from the cell. Other hydrogenosomal enzymes also have similar amino-terminal extensions which probably play a role in organellar targeting and translocation of the newly synthesized polypeptides. A comparison of 19 related enzymes from bacteria and eukaryotes with the maeA product revealed 34–45 percent amino acid identity. Phylogenetic reconstruction based on nonconservative amino acid differences with maximum parsimony (phylogenetic analysis using parsimony, PAUP) and distance based (neighbor-joining, NJ) methods showed that the T. vaginalis enzyme is the most divergent of all eukaryotic malic enzymes, indicating its long independent evolutionary history.     

Flagellar apparatus and nuclear chambers of the green dinoflagellate Gymnodinium chlorophorum

The green dinoflagellate Gymnodinium chlorophorum (BAH ME 100, the type culture) was reexamined with emphasis on the structure of the flagellar apparatus and nuclear envelope. Like other Gymnodinium species, G. chlorophorum possessed a nuclear fibrous connective linking the flagellar apparatus and the nucleus, albeit in a very reduced and unique form. Microtubules nucleated from the R3 flagellar root associated with the nuclear fibrous connective and terminated at the nucleus, a novel arrangement not known in any other dinoflagellate. Although overlooked by previous researchers, nuclear chambers were present in G. chlorophorum similar to those reported in Gymnodinium aureolum and Gymnodinium nolleri. In contrast to the type species of Gymnodinium, Gymnodinium fuscum, only one nuclear pore was present per chamber. The presence of a feeding tube (peduncle) suggests that G. chlorophorum is mixotrophic. Although the fine structure of G. chlorophorum revealed its affiliation to the Gymnodinium group the above discrepancies set it apart, indicating that it might belong in a different genus.     

Reconstruction of the flagellar apparatus in Ploeotia costata (Euglenozoa) and its relationship to other euglenoid flagellar apparatuses

The flagellar apparatus of Ploeotia costata Farmer and Triemer was reconstructed using serial sectioning and TEM. The flagellar apparatus is similar to other euglenoids having two flagella arising from basal bodies connected by a striated fiber, and three asymmetrically arranged roots. The flagella emerge subapically from between the two ventral pellicle strips. The dorsal flagellum is 1/2 the body length and actively pulls the cell, while the ventral flagellum is twice the body length and drags along the substrate surface. The ventral and dorsal roots are on the opposite sides of their respective basal bodies, while the intermediate root is associated with the ventral flagellum on the side closest to the dorsal basal body. The dorsal root lines the dorsal side of the reservoir and after giving rise to the dorsal band lines the right side of the reservoir/canal. The ventral and intermediate roots join at the reservoir forming the intermediate-ventral root, which lines the left and ventral sides of the reservoir/canal. There was no evidence of a microtubule-reinforced pocket in P. costata. Comparisons with Ploeotia vilrea, Lentomonas applanatum, and related flagellar apparatuses led to the conclusion that the basic euglenoid flagellar structure is symplesiomorphic but with enough variation to be taxonomically diagnostic.     

Ultrastructure of the flagellar apparatus in the green flagellateSpermatozopsis similis

The ultrastructure of the flagellar apparatus of the naked, biflagellate green algaSpermatozopsis similis Preisig & Melkonian has been studied in detail using an absolute configuration analysis. The two basal bodies are displaced by 350 nm in the 1/7 o'clock direction and do not overlap proximally. They are interconnected by a principal distal connecting fibre consisting of a bundle of 5–8 nm filaments and possibly two proximal striated connecting fibres. The flagellar root system is cruciate (5-2-5-2 or 4-2-4-2 system) and contains a prominent continuous system I fibre overlying the two opposite two-stranded roots. A system II fibre is absent. Pronounced structural differences have been observed in the flagellar apparatus ultrastructure at two types of flagella orientation: During backward swimming basal bodies are parallel, the distal connecting fibre is extremely contracted; during forward swimming basal bodies assume various angles (from 20° to 180°) and the connecting fibre is about five times longer compared to the contracted state. The function of the connecting fibre as a contractile organelle and the mechanism of its contraction are discussed. On the basis of the flagellar apparatus ultrastructure,Spermatozopsis similis is related toChlamydomonas-type green algae.     

The flagellar apparatus and cytoskeleton of the dinoflagellates

Summary Modern microscopical approaches have allowed more accurate investigations of the three-dimensional nature of the dinoflagellate flagellar apparatus (FA) and several other cytoskeletal protein complexes. Our presentation overviews the nature of the dinoflagellate FA and cytoskeleton in a number of taxa and compares them with those of other protists. As with other protists, the FA of the dinoflagellates can be characterized by the presence of fibrous and microtubular components. Our studies and others indicate that the dinoflagellate FA can be expected to possess a striated fibrous root on the basal body of the transverse flagellum and a multimembered microtubular root on the basal body of the longitudinal flagellum. Two other features that appear widespread in the group are the transverse striated root associated microtubule (tsrm) and the transverse microtubular root (tmr). The tsrm extends at least half the length of the transverse striated root while the tmr extends from the transverse basal body toward the exit aperture of the transverse flagellum. In most cases, the tmr gives rise to several cytoplasmic microtubules at a right angle. The apparent conserved nature of these roots leads us to the conclusion that the dinoflagellate FA can be compared to the FA of the cryptomonads, chrysophytes, and the ciliates for phylogenetic purposes. Of these groups, the chrysophytes possess an FA with the most structures in common with the dinoflagellates. Our immunomicroscopical investigations of the microtubular, actin and centrin components of the dinoflagellate cytoskeleton point to the comparative usefulness of these cytological features.Abbreviations aptb apical transverse microtubular band - FA flagellar apparatus - Imr longitudinal microtubular root - mls multilayered structure - tmr transverse microtubular root - tmre transverse microtubular root extension - tsr transverse striated fibrous root - tsrm transverse striated root associated microtubule     

The flagellar apparatus of the glaucophyte Cyanophora cuspidata

Glaucophytes are a kingdom‐scale lineage of unicellular algae with uniquely underived plastids. The genus Cyanophora is of particular interest because it is the only glaucophyte that is a flagellate throughout its life cycle, making its morphology more directly comparable than other glaucophytes to other eukaryote flagellates. The ultrastructure of Cyanophora has already been studied, primarily in the 1960s and 1970s. However, the usefulness of that work has been undermined by its own limitations, subsequent misinterpretations, and a recent taxonomic revision of the genus. For example, Cyanophora's microtubular roots have been widely reported as cruciate, with rotationally symmetrical wide and thin roots, although the first ultrastructural work described it as having three wide and one narrow root. We examine Cyanophora cuspidata using scanning and transmission electron microscopy, and construct a model of its cytoskeleton using serial‐section TEM. We confirm the earlier model, with asymmetric roots. We describe previously unknown and unsuspected features of its microtubular roots, including (i) a rearrangement of individual microtubules within the posterior right root, (ii) a splitting of the posterior left root into two subroots, and (iii) the convergence and termination of the narrow roots against wider ones in both the anterior and posterior subsystems of the flagellar apparatus. We also describe a large complement of nonmicrotubular components of the cytoskeleton, including a substantial connective between the posterior right root and the anterior basal body. Our work should serve as the starting point for a re‐examination of both internal glaucophyte diversity and morphological evolution in eukaryotes.     

Primary structure and eubacterial relationships of the pyruvate:Ferredoxin oxidoreductase of the amitochondriate eukaryoteTrichomonas vaginalis

In the eukaryotic unicellular organismTrichomonas vaginalis a key step of energy metabolism, the oxidative decarboxylation of pyruvate with the formation of acetyl-CoA, is catalyzed by the iron-sulfur protein pyruvate:ferredoxin oxidoreductase (PFO) and not by the almost-ubiquitous pyruvate dehydrogenase multienzyme complex. This enzyme is localized in the hydrogenosome, an organelle bounded by a double membrane. PFO and its closely related homolog, pyruvate: flavodoxin oxidoreductase, are enzymes found in a number of archaebacteria and eubacteria. The presence of these enzymes in eukaryotes is restricted, however, to a few amitochondriate groups. To gain more insight into the evolutionary relationships ofT. vaginalis PFO we determined the primary structure of its two genes (pfoA andpfoB). The deduced amino acid sequences showed 95% positional identity. Motifs implicated in related enzymes in liganding the Fe-S centers and thiamine pyrophosphate were well conserved. TheT. vaginalis PFOs were found to be homologous to eubacterial pyruvate: flavodoxin oxidoreductases and showed about 40% amino acid identity to these enzymes over their entire length. Lack of eubacterial PFO sequences precluded a comparison.pfoA andpfoB revealed a greater distance from related enzymes of Archaebacteria. The conceptual translation of the nucleotide sequences predicted an amino-terminal pentapeptide not present in the mature protein. This processed leader sequence was similar to but shorter than leader sequences noted in other hydrogenosomal proteins. These sequences are assumed to be involved in organellar targeting and import. The results underscore the unusual characteristics ofT. vaginalis metabolism and of their hydrogenosomes. They also suggest that in its energy metabolismT. vaginalis is closer to eubacteria than archaebacteria.Abbreviations PCR DNA polymerase chain reaction - PDH pyruvate dehydrogenase - PFO pyruvate:ferredoxin oxidoreductase - TPP thiamine pyrophosphate Correspondence to: M. Müller     

The Evolution of the Vahlkampfiidae as Deduced from 16S-like Ribosomal RNA Analysis

ABSTRACT. The amoebae, a phenotypically diverse, paraphyletic group of protists, have been largely neglected by molecular phy-logeneticists. To better understand the evolution of amoebae, we sequenced and analyzed the 16S-like ribosomal RNA genes of three vahlkampfiid amoebae: Paratetramitus jugosus, Tetramitus rostratus and Vahlkampfia lobospinosa . The Vahlkampfiidae lineage is monophyletic, branches early along the eukaryotic line of descent, and is not a close relative of the multicellular amoebae that also reversibly transform from amoebae to flagellates.     

The flagellar apparatus ofMesostigma viride (Prasinophyceae): Multilayered structures in a scaly green flagellate

Mesostigma viride Lauterborn (Prasinophyceae) is the first green flagellate found to have multilayered structures (MLS) in its flagellar apparatus. MLS's were previously known from green algae only in charophycean swarmers, linking theCharophyceae to the origin of land plants, whose male gametes (when flagellated) also possess an MLS.M. viride is, therefore, probably more closely related to the origin of theCharophyceae than any other green flagellate that has been thoroughly studied so far. The occurrence of MLS's in green flagellates and apparently in other algae and protozoans suggests that an MLS occurred in an ancient group of flagellates and has survived in various protistan lines, including the line of green algae related to land plants. The occurrence of a synistosome inM. viride and other of its characteristics suggest that it is more closely related toPyramimonas than to other genera of scaly green flagellates.This work was supported by National Science Foundation Grant DEB-78-03554.     

The endosymbiotic origin,diversification and fate of plastids

Plastids and mitochondria each arose from a single endosymbiotic event and share many similarities in how they were reduced and integrated with their host. However, the subsequent evolution of the two organelles could hardly be more different: mitochondria are a stable fixture of eukaryotic cells that are neither lost nor shuffled between lineages, whereas plastid evolution has been a complex mix of movement, loss and replacement. Molecular data from the past decade have substantially untangled this complex history, and we now know that plastids are derived from a single endosymbiotic event in the ancestor of glaucophytes, red algae and green algae (including plants). The plastids of both red algae and green algae were subsequently transferred to other lineages by secondary endosymbiosis. Green algal plastids were taken up by euglenids and chlorarachniophytes, as well as one small group of dinoflagellates. Red algae appear to have been taken up only once, giving rise to a diverse group called chromalveolates. Additional layers of complexity come from plastid loss, which has happened at least once and probably many times, and replacement. Plastid loss is difficult to prove, and cryptic, non-photosynthetic plastids are being found in many non-photosynthetic lineages. In other cases, photosynthetic lineages are now understood to have evolved from ancestors with a plastid of different origin, so an ancestral plastid has been replaced with a new one. Such replacement has taken place in several dinoflagellates (by tertiary endosymbiosis with other chromalveolates or serial secondary endosymbiosis with a green alga), and apparently also in two rhizarian lineages: chlorarachniophytes and Paulinella (which appear to have evolved from chromalveolate ancestors). The many twists and turns of plastid evolution each represent major evolutionary transitions, and each offers a glimpse into how genomes evolve and how cells integrate through gene transfers and protein trafficking.     

Andalucia (n. gen.)--the deepest branch within jakobids (Jakobida; Excavata), based on morphological and molecular study of a new flagellate from soil

A new heterotrophic flagellate (Andalucia godoyi n. gen. n. sp.) is described from soil. Earlier preliminary 18S rRNA analyses had indicated a relationship with the phylogenetically difficult-to-place jakobid Jakoba incarcerata. Andalucia godoyi is a small (3-5 mum) biflagellated cell with a ventral feeding groove. It has tubular mitochondrial cristae. There are two major microtubular roots (R1, R2) and a singlet root associated with basal body 1 (posterior). The microtubular root R1 is associated with non-microtubular fibres "I,"B," and "A," and divides in two parts, while R2 is associated with a "C" fibre. These structures support the anterior portion of the groove. Several features of A. godoyi are characteristic of jakobids: (i) there is a single dorsal vane on flagellum 2; (ii) the C fibre has the jakobid multilaminate substructure; (iii) the dorsal fan of microtubules originates in very close association with basal body 2; and (iv) there is no "R4" microtubular root associated with basal body 2. Morphological analyses incorporating the A. godoyi data strongly support the monophyly of all jakobids. Our 18S rRNA phylogenies place A. godoyi and J. incarcerata as a strong clade, which falls separately from other jakobids. Statistical tests do not reject jakobid monophyly, but a specific relationship between Jakoba libera and J. incarcerata and/or A. godoyi is rejected. Therefore, we have established a new genus Andalucia n. gen. with the type species Andalucia godoyi n. sp., and transfer Jakoba incarcerata to Andalucia as Andalucia incarcerata n. comb.     

The genetic diversity of planktic foraminifera and the global distribution of ribosomal RNA genotypes

Fossil planktic foraminifers in the ocean sediments play an unparalleled role in our understanding of the oceanographic environment in the past. An in depth knowledge of their diversity, ecology and biogeography in the modern ocean lies central to the interpretation of the fossil assemblages. In comparison with their benthic counterparts, planktic foraminifera have a very limited diversity of around fifty extant morphospecies. Their morphospecies diversity peaks in the sub-tropics and decreases steeply towards the poles. Traditional species concepts have partitioned morphological types into distinct species (morphospecies) based on test shape, but genetic studies show that individual morphospecies are actually complexes of several discrete genetic types (genotypes). Many of these genotypes have distinct ecologies and novel adaptations that are consistent with species-level classification, indicating that the true diversity of planktic foraminifers has been greatly underestimated. Although planktic foraminifera are clearly capable of long-distance dispersal, they may be constrained by both physical and ecological barriers that vary according to the evolutionary history and ecology of the individual genotypes within a morphospecies. These differences lead to diverse biogeographies. Here, we provide an overview of the genetic and biogeographic data available to date for the planktic foraminifera and present global biogeographies highlighting the distribution of genetic types in the eight planktic foraminiferal morphospecies for which detailed molecular evidence is available.     

Parvularia atlantis gen. et sp. nov., a Nucleariid Filose Amoeba (Holomycota,Opisthokonta)

The opisthokonts constitute a eukaryotic supergroup divided into two main clades: the holozoans, which include animals and their unicellular relatives, and the holomycotans, which include fungi, opisthosporidians, and nucleariids. Nucleariids are phagotrophic filose amoebae that phenotypically resemble more their distant holozoan cousins than their holomycotan phylogenetic relatives. Despite their evolutionary interest, the diversity and internal phylogenetic relationships within the nucleariids remain poorly studied. Here, we formally describe and characterize by molecular phylogeny and microscopy observations Parvularia atlantis gen. et sp. nov. (formerly Nuclearia sp. ATCC 50694), and compare its features with those of other nucleariid genera. Parvularia is an amoebal genus characterized by radiating knobbed and branching filopodia. It exhibits prominent vacuoles observable under light microscopy, a cyst‐like stage, and completely lacks cilia. P. atlantis possesses one or two nuclei with a central nucleolus, and mitochondria with flat or discoid cristae. These morphological features, although typical of nucleariids, represent a combination of characters different to those of any other described Nuclearia species. Likewise, 18S rRNA‐based phylogenetic analyses show that P. atlantis represents a distinct lineage within the nucleariids.