Rhizarian 'Novel Clade 10' Revealed as Abundant and Diverse Planktonic and Terrestrial Flagellates, including Aquavolon n. gen.

Rhizarian 'Novel Clade 10' (NC10) is frequently detected by 18S rRNA gene sequencing studies in freshwater planktonic samples. We describe a new genus and two species of eukaryovorous biflagellate protists, Aquavolon hoantrani n. gen. n. sp. and A. dientrani n. gen. n. sp., which represent the first morphologically characterized members of NC10, here named Aquavolonida ord. nov. The slightly metabolic cells possess naked heterodynamic flagella, whose kinetosomes lie at a right angle to each other and are connected by at least one fibril. Unlike their closest known relative Tremula longifila, they rotate around their longitudinal axis when swimming and only very rarely glide on surfaces. Screening of a wide range of environmental DNA extractions with lineage-specific PCR primers reveals that Aquavolonida consists of a large radiation of protists, which are most diversified in freshwater planktonic habitats and as yet undetected in marine environments. Earlier-branching lineages in Aquavolonida include less frequently detected organisms from soils and freshwater sediments. The 18S rRNA gene phylogeny suggests that Aquavolonida forms a common evolutionary lineage with tremulids and uncharacterized 'Novel Clade 12', which likely represents one of the deepest lineages in the Rhizaria, separate from Cercozoa (Filosa), Endomyxa, and Retaria.

Colorful seashells: Identification of haem pathway genes associated with the synthesis of porphyrin shell color in marine snails.

Very little is known about the evolution of molluskan shell pigments, although Mollusca is a highly diverse, species rich, and ecologically important group of animals comprised of many brightly colored taxa. The marine snail genus Clanculus was chosen as an exceptional model for studying the evolution of shell color, first, because in Clanculus margaritarius and Clanculus pharaonius both shell and foot share similar colors and patterns; and second, because recent studies have identified the pigments, trochopuniceus (pink-red), and trochoxouthos (yellow-brown), both comprised of uroporphyrin I and uroporphyrin III, in both shell and colored foot tissue of these species. These unusual characteristics provide a rare opportunity to identify the genes involved in color production because, as the same pigments occur in the shell and colored foot tissue, the same color-related genes may be simultaneously expressed in both mantle (which produces the shell) and foot tissue. In this study, the transcriptomes of these two Clanculus species along with a third species, Calliostoma zizyphinum, were sequenced to identify genes associated with the synthesis of porphyrins. Calliostoma zizyphinum was selected as a negative control as trochopuniceus and trochoxouthos were not found to occur in this species. As expected, genes necessary for the production of uroporphyrin I and III were found in all three species, but gene expression levels were consistent with synthesis of uroporphyrins in mantle and colored foot tissue only in Clanculus. These results are relevant not only to understanding the evolution of shell pigmentation in Clanculus but also to understanding the evolution of color in other species with uroporphyrin pigmentation, including (mainly marine) mollusks soft tissues and shells, annelid and platyhelminth worms, and some bird feathers.

Morphology, ultrastructure, and small subunit rDNA phylogeny of the marine heterotrophic flagellate Goniomonas aff. amphinema.

Marine goniomonads have a worldwide distribution but ultrastructural information has not been available so far. An isolate of the heterotrophic marine nanoflagellate Goniomonas (G. aff. amphinema) from North Wales (UK) has been studied, providing information on its morphology and cellular structure using video, electron, laser scanning confocal microscopy (LSCM), and atomic force microscopy. Here, we describe a new feature, a granular area, potentially involved in particle capture and feeding. The binding of the lectin wheat germ agglutinin to the granular area of cells with discharged ejectisomes indicates the adhesive nature of this novel feature. The presence of a microtubular intracellular cytopharynx, apparently also used for feeding, has been revealed by LSCM. The small subunit rRNA gene of the isolate has been sequenced (1,788 bp). Phylogenetic results corroborate significant genetic divergence within the marine members of Goniomonas. This work highlights the need for integrated morphological, ultrastructural, and molecular investigation when describing and studying heterotrophic nanoflagellates.

A molecular perspective on ecological differentiation and biogeography of cyclotrichiid ciliates.

Cyclotrichiids are of ecological and evolutionary interest by virtue of their importance in red tide formation, their highly divergent small subunit (SSU) ribosomal RNA (rRNA) genes, kleptoplastidy, and utility as indicators of eutrophication. However, only seven strains have had their SSU rRNA genes sequenced and their environmental diversity and distribution are largely unknown. We probed 67 globally dispersed freshwater column/sediment and soil DNA samples (eDNAs) and constructed 24 environmental gene libraries using polymerase chain reaction primers specific to an uncharacterised cyclotrichiid subgroup. We reveal a novel, globally ubiquitous freshwater clade comprising 25 genetically distinct SSU ribosomal DNA (rDNA) sequences (SSU-types). Some identical SSU-types were detected at globally widely distributed sites. The SSU-types form four distinct phylogenetic clusters according to marine or non-marine provenance, suggesting at least one major marine-freshwater evolutionary transition within the cyclotrichiids. We used the same primers to sample intensively 18 sampling points in 13 closely situated lakes, each characterised by 14 environmental variables, and showed that molecular detection or non-detection of cyclotrichiids was most significantly influenced by levels of total phosphorus, dissolved organic carbon, and chlorophyll a. Within the subset of lakes in which cyclotrichiids were detected, closely related SSU-types differed in their ecological preferences to pH, total phosphorus, and sample depth.

Classification of the peritrich ciliate Opisthonecta matiensis (Martín-Cereceda et al. 1999) as Telotrochidium matiense nov. comb., based on new observations and SSU rDNA phylogeny.

New observations on Opisthonecta matiensis Martín-Cereceda et al. [1999. Description of Opisthonecta matiensis n. sp. (Protozoa, Ciliophora), a new peritrich ciliate from wastewater. J. Eukaryot. Microbiol. 46, 283-289] especially the lack of an epistomial membrane, reveal that the species does not belong to the genus Opisthonecta, but to Telotrochidium, the other genus within the family Opisthonectidae Foissner, 1975. The contractile vacuole and the cytopyge are on the dorsal wall of the vestibulum and the trochal band is limited distally and proximally by rows of narrowly spaced pellicular pores. Thus the species is redefined as Telotrochidium matiense nov. comb. The morphological, cortical and nuclear events occurring during conjugation are illustrated, compared with those in other species, and phylogenetically discussed. Invariably, the microconjugants attach to and penetrate the lateral side of the macroconjugants. Nuclear processes are very similar to those reported from other peritrichs. The small subunit rRNA gene (SSU rDNA) is sequenced and the phylogeny within Opisthonectidae and peritrichs examined. T. matiense is more closely related to Epistylis (63% Maximum Parsimony (MP), 85% Maximum Likelihood (ML)) than to any other genus, while another representative of the family, viz., Opisthonecta henneguyi, is closely related to Vorticella microstoma, Astylozoon enriquesi and clone RT3n18 (100% MP, 100% ML). Morphology and gene sequences suggest that Telotrochidium and Opisthonecta have derived from different lineages of stalked peritrichs: Opisthonecta could have arisen from peritrichs with stalk myonemes, while Telotrochidium probably evolved from peritrichs without stalk myonemes.

A genomic survey of the fish parasite Spironucleus salmonicida indicates genomic plasticity among diplomonads and significant lateral gene transfer in eukaryote genome evolution.

BACKGROUND: Comparative genomic studies of the mitochondrion-lacking protist group Diplomonadida (diplomonads) has been lacking, although Giardia lamblia has been intensively studied. We have performed a sequence survey project resulting in 2341 expressed sequence tags (EST) corresponding to 853 unique clones, 5275 genome survey sequences (GSS), and eleven finished contigs from the diplomonad fish parasite Spironucleus salmonicida (previously described as S. barkhanus). RESULTS: The analyses revealed a compact genome with few, if any, introns and very short 3' untranslated regions. Strikingly different patterns of codon usage were observed in genes corresponding to frequently sampled ESTs versus genes poorly sampled, indicating that translational selection is influencing the codon usage of highly expressed genes. Rigorous phylogenomic analyses identified 84 genes--mostly encoding metabolic proteins--that have been acquired by diplomonads or their relatively close ancestors via lateral gene transfer (LGT). Although most acquisitions were from prokaryotes, more than a dozen represent likely transfers of genes between eukaryotic lineages. Many genes that provide novel insights into the genetic basis of the biology and pathogenicity of this parasitic protist were identified including 149 that putatively encode variant-surface cysteine-rich proteins which are candidate virulence factors. A number of genomic properties that distinguish S. salmonicida from its human parasitic relative G. lamblia were identified such as nineteen putative lineage-specific gene acquisitions, distinct mutational biases and codon usage and distinct polyadenylation signals. CONCLUSION: Our results highlight the power of comparative genomic studies to yield insights into the biology of parasitic protists and the evolution of their genomes, and suggest that genetic exchange between distantly-related protist lineages may be occurring at an appreciable rate in eukaryote genome evolution.

Hydrogenosomal succinyl-CoA synthetase from the rumen-dwelling fungus Neocallimastix patriciarum; an energy-producing enzyme of mitochondrial origin.

Hydrogenosomes are hydrogen-producing organelles that are related to mitochondria and found in a variety of evolutionarily unrelated anaerobic microbial eukaryotes. Similar to classic mitochondria, hydrogenosomes contain the enzyme catalyzing the only reaction of the citric acid cycle directly producing energy; succinyl-CoA synthetase. We have isolated and characterized the genes encoding both subunits of this enzyme from the anaerobic chytrid fungus Neocallimastix patriciarum, a model organism in hydrogenosome research. Both subunits contain all characteristic features of this enzyme, including predicted hydrogenosomal targeting signals. Phylogenetic analyses of succinyl-CoA synthetase clearly indicate its mitochondrial ancestry, both by affiliation with mitochondrially localized fungal homologues and by the sisterhood of the eukaryotic succinyl-CoA synthetase clade with alpha-proteobacteria. Our analyses of the Trichomonas vaginalis SCS sequences also confirmed the mitochondrial affiliation of these hydrogenosomal enzymes, in contrast to previous results. While both hydrogenosomal and mitochondrial succinyl-CoA synthetase homologues have been identified, no succinyl-CoA synthetase proteins were identifiable in taxa possessing another mitochondrially derived organelle, the mitosome. Our analyses further confirm the mitochondrial ancestry of the Neocallimastix hydrogenosome and sheds light upon the stepwise process by which mitochondria evolve into alternate forms of the organelle.

Inference of the phylogenetic position of oxymonads based on nine genes: support for metamonada and excavata.

Circumscribing major eukaryote groups and resolving higher order relationships between them are among the most challenging tasks facing molecular evolutionists. Recently, evidence suggesting a new supergroup (the Excavata) comprising a wide array of flagellates has been collected. This group consists of diplomonads, retortamonads, Carpediemonas, heteroloboseans, Trimastix, jakobids, and Malawimonas, all of which possess a particular type of ventral feeding groove that is proposed to be homologous. Euglenozoans, parabasalids, and oxymonads have also been associated with Excavata as their relationships to one or more core excavate taxa were demonstrated. However, the main barrier to the general acceptance of Excavata is that its existence is founded primarily on cytoskeletal similarities, without consistent support from molecular phylogenetics. In gene trees, Excavata are typically not recovered together. In this paper, we present an analysis of the phylogenetic position of oxymonads (genus Monocercomonoides) based on concatenation of eight protein sequences (alpha-tubulin, beta-tubulin, gamma-tubulin, EF-1alpha, EF-2, cytosolic (cyt) HSP70, HSP90, and ubiquitin) and 18S rRNA. We demonstrate that the genes are in conflict regarding the position of oxymonads. Concatenation of alpha- and beta-tubulin placed oxymonads in the plant-chromist part of the tree, while the concatenation of other genes recovered a well-supported group of Metamonada (oxymonads, diplomonads, and parabasalids) that branched weakly with euglenozoans--connecting all four excavates included in the analyses and thus providing conditional support for the existence of Excavata.

Hydrogenosomes, mitochondria and early eukaryotic evolution.

Available data suggest that unusual organelles called hydrogenosomes, that make ATP and hydrogen, and which are found in diverse anaerobic eukaryotes, were once mitochondria. The evolutionary origins of the enzymes used to make hydrogen, pyruvate:ferredoxin oxidoreductase (PFO) and hydrogenase, are unresolved, but it seems likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobes, these proteins are found in diverse eukaryotic cells, including our own, where they are targeted to different cell compartments. Organelles related to mitochondria and hydrogenosomes have now been found in species of anaerobic and parasitic protozoa that were previously thought to have separated from other eukaryotes before the mitochondrial endosymbiosis. Thus it is possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, bearing testimony to the important role that the mitochondrial endosymbiosis has played in eukaryotic evolution. It remains to be seen if members of this family of organelles share a common function essential to the eukaryotic cell, that provides the underlying selection pressure for organelle retention under different living conditions.

Fungal hydrogenosomes contain mitochondrial heat-shock proteins.

At least three groups of anaerobic eukaryotes lack mitochondria and instead contain hydrogenosomes, peculiar organelles that make energy and excrete hydrogen. Published data indicate that ciliate and trichomonad hydrogenosomes share common ancestry with mitochondria, but the evolutionary origins of fungal hydrogenosomes have been controversial. We have now isolated full-length genes for heat shock proteins 60 and 70 from the anaerobic fungus Neocallimastix patriciarum, which phylogenetic analyses reveal share common ancestry with mitochondrial orthologues. In aerobic organisms these proteins function in mitochondrial import and protein folding. Homologous antibodies demonstrated the localization of both proteins to fungal hydrogenosomes. Moreover, both sequences contain amino-terminal extensions that in heterologous targeting experiments were shown to be necessary and sufficient to locate both proteins and green fluorescent protein to the mitochondria of mammalian cells. This finding, that fungal hydrogenosomes use mitochondrial targeting signals to import two proteins of mitochondrial ancestry that play key roles in aerobic mitochondria, provides further strong evidence that the fungal organelle is also of mitochondrial ancestry. The extraordinary capacity of eukaryotes to repeatedly evolve hydrogen-producing organelles apparently reflects a general ability to modify the biochemistry of the mitochondrial compartment.

Mitochondria and hydrogenosomes are two forms of the same fundamental organelle.

Published data suggest that hydrogenosomes, organelles found in diverse anaerobic eukaryotes that make energy and hydrogen, were once mitochondria. As hydrogenosomes generally lack a genome, the conversion is probably one way. The sources of the key hydrogenosomal enzymes, pyruvate : ferredoxin oxidoreductase (PFO) and hydrogenase, are not resolved by current phylogenetic analyses, but it is likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobic eukaryotes, the proteins are intimately integrated into the fabric of diverse eukaryotic cells, where they are targeted to different cell compartments, and not just hydrogenosomes. There is no evidence supporting the view that PFO and hydrogenase originated from the mitochondrial endosymbiont, as posited by the hydrogen hypothesis for eukaryogenesis. Other organelles derived from mitochondria have now been described in anaerobic and parasitic microbial eukaryotes, including species that were once thought to have diverged before the mitochondrial symbiosis. It thus seems possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, to which different types of biochemistry can be targeted. It remains to be seen if, despite their obvious differences, this family of organelles shares a common function of importance for the eukaryotic cell, other than energy production, that might provide the underlying selection pressure for organelle retention.

Conserved properties of hydrogenosomal and mitochondrial ADP/ATP carriers: a common origin for both organelles.

Mitochondria are one of the hallmarks of eukaryotic cells, exporting ATP in exchange for cytosolic ADP using ADP/ATP carriers (AAC) located in the inner mitochondrial membrane. In contrast, several evolutionarily important anaerobic eukaryotes lack mitochondria but contain hydrogenosomes, peculiar organelles of controversial ancestry that also supply ATP but, like some fermentative bacteria, make molecular hydrogen in the process. We have now identified genes from two species of the hydrogenosome-containing fungus Neocallimastix that have three-fold sequence repeats and signature motifs that, along with phylogenetic analysis, identify them as AACs. When expressed in a mitochondrial AAC- deficient yeast strain, the hydrogenosomal protein was correctly targeted to the yeast mitochondria inner membrane and yielded mitochondria able to perform ADP/ATP exchange. Characteristic inhibitors of mitochondrial AACs blocked adenine nucleotide exchange by the Neocallimastix protein. Thus, our data demonstrate that fungal hydrogenosomes and yeast mitochondria use the same pathway for ADP/ATP exchange. These experiments provide some of the strongest evidence yet that yeast mitochondria and Neocallimastix hydrogenosomes are but two manifestations of the same fundamental organelle.