New phagotrophic euglenoid species (new genus Decastava; Scytomonas saepesedens; Entosiphon oblongum), Hsp90 introns, and putative euglenoid Hsp90 pre-mRNA insertional editing.

We describe three new phagotrophic euglenoid species by light microscopy and 18S rDNA and Hsp90 sequencing: Scytomonas saepesedens; Decastava edaphica; Entosiphon oblongum. We studied Scytomonas and Decastava ultrastructure. Scytomonas saepesedens feeds when sessile with actively beating cilium, and has five pellicular strips with flush joints and Calycimonas-like microtubule-supported cytopharynx. Decastava, sister to Keelungia forming new clade Decastavida on 18S rDNA trees, has 10 broad strips with cusp-like joints, not bifurcate ridges like Ploeotia and Serpenomonas (phylogenetically and cytologically distinct genera), and Serpenomonas-like feeding apparatus (8-9 unreinforced microtubule pairs loop from dorsal jaw support to cytostome). Hsp90 and 18S rDNA trees group Scytomonas with Petalomonas and show Entosiphon as the earliest euglenoid branch. Basal euglenoids have rigid longitudinal strips; derived clade Spirocuta has spiral often slideable strips. Decastava Hsp90 genes have introns. Decastava/Entosiphon Hsp90 frameshifts imply insertional RNA editing. Petalomonas is too heterogeneous in pellicle structure for one genus; we retain Scytomonas (sometimes lumped with it) and segregate four former Petalomonas as new genus Biundula with pellicle cross section showing 2-8 smooth undulations and typified by Biundula (=Petalomonas) sphagnophila comb. n. Our taxon-rich site-heterogeneous rDNA trees confirm that Heteronema is excessively heterogeneous; therefore we establish new genus Teloprocta for Heteronema scaphurum.

Novel cultured protists identify deep-branching environmental DNA clades of cercozoa: New Genera Tremula, Micrometopion, Minimassisteria, Nudifila, Peregrinia.

We describe three new orders of filosan Cercozoa, five new deep-branching genera, eight new species of Thaumatomonas, Reckertia, Spongomonas, Rhogostoma, Agitata, Neoheteromita and Paracercomonas, sequence their 18S rDNA, and construct 18S rDNA trees for 148 Cercozoa. Our phylogeny indicates that Filosa were ancestrally gliding flagellates; non-flagellate filose amoebae evolved from them five times independently. The new genera are more closely related to environmental DNA sequences than cultured organisms. Tremula longifila, a zooflagellate glider on both flagella (unlike other Cercozoa), is the most divergent filosan (Tremulida ord. n.). Micrometopion nutans is a eukaryote-eating gliding zooflagellate like Metopion and Metromonas. Minimassisteria diva is a widespread trimorphic marine amoeboflagellate granofilosan. Peregrinia clavideferens, a non-testate, scale-bearing, filose amoeba, branches deeply in Thaumatomonadida, which are probably sisters to Spongomonadida. Nudifila producta is a filose amoeboflagellate related to Clautriavia and Marimonadida (ord. n., e.g. Pseudopirsonia, Auranticordis). We substantially revise Imbricatea, now including Spongomonadida, and Thecofilosea to include Phaeodaria. Thecofilosea and Imbricatea and Thecofilosea are sisters, both arguably ancestrally rigid gliding flagellates with ventral pseudopod-emitting grooves. Scale-free Ovulinata parva is sister to Paulinella, so imbricate silica scales can be lost. Internal hollow silica skeletons evolved twice in Thecofilosea (Ebriida, Phaeodaria) or were multiply lost. Protaspa replaces preoccupied 'Protaspis'.

"Not a very nice subject." Changing views of parasites and parasitology in the twentieth century.

The man in-the-street who frequently asks the question "Why am I here?" finds even more difficulty with the question "Why are parasites here?" The public's distaste for parasites (and by implication, for parasitologists!) is therefore understandable, as maybe was the feeling of early 20th century biologists that parasites were a puzzle because they did not conform to the then widely held association between evolution and progress, let alone the reason why a benevolent Creator should have created them. In mid-century, the writer, contemplating a career in parasitology was taken aback when he found that extolled contemporary biologists disdained parasites or thought little of parasitology as an intellectual subject. These attitudes reflected a lack of appreciation of the important role of parasites in generating evolutionary novelty and speciation, also unawareness of the value of parasite life-cycle studies for formulating questions of wider significance in biology, deficiencies which were gratifyingly beginning to be remedied in the latter half of the century.

Phylogeny and classification of Cercomonadida (Protozoa, Cercozoa): Cercomonas, Eocercomonas, Paracercomonas, and Cavernomonas gen. nov.

Cercomonads (=Cercomonadida) are biflagellate gliding bacterivorous protozoa, abundant and diverse in soil and freshwater. We establish 56 new species based on 165 cultures, differential interference contrast microscopy, and 18S and ITS2 rDNA sequencing, and a new genus Cavernomonas studied by scanning electron microscopy. We fundamentally revise the phylogeny and classification of cercomonad Cercozoa. We describe 40 Cercomonas species (35 novel), six Eocercomonas (five novel), two Cavernomonas, and 18 Paracercomonas species (14 novel). We obtained additional cercomonad clade A (Cercomonas, Eocercomonas, Cavernomonas) sequences from multiple environmental DNA libraries. The most commonly cultivated genotypes are not the commonest in environmental DNA, suggesting that cercomonad ecology is far more complex than implied by laboratory cultures. Cercomonads have never been isolated from saline environments, although some species can grow in semi-saline media in the laboratory, and environmental DNA libraries regularly detect them in coastal marine sediments. The first ultrastructural study of an anaerobic cercozoan, Paracercomonas anaerobica sp. nov., a highly divergent cercomonad, shows much simpler ciliary roots than in clade A cercomonads, a ciliary hub-lattice and axosome, and mitochondria with tubular cristae, consistent with it being only facultatively anaerobic. We also describe Agitata tremulans gen. et sp. nov., previously misidentified as Cercobodo (=Dimastigamoeba) agilis Moroff.

Phylogeny, taxonomy, and astounding genetic diversity of glissomonadida ord. nov., the dominant gliding zooflagellates in soil (Protozoa: Cercozoa).

The cercozoan family Heteromitidae comprises morphologically rather uniform gliding zooflagellates, including Bodomorpha and Heteromita, the most ubiquitous and numerous soil protozoa. The generally used name 'Heteromita globosa' for the commonest gliding biflagellates is incorrect. 'Heteromita' Dujardin, 1841 originally contained only two probable euglenozoans and an unidentifiable flagellate, making it inapplicable to Cercozoa. Accordingly, we establish a new order Glissomonadida for Heteromitidae sensu Cavalier-Smith and Chao, 2003. We cultured over 100 glissomonad strains, sequenced their 18S rRNA genes, and studied their behaviour and morphology by differential interference contrast high definition video microscopy. Group-specific amplification and sequencing of over 450 18S rRNA genes from environmental DNA shows that one temperate grassland plot has hundreds of species, there are thousands globally, and tropical species often differ. Glissomonads are probably sisters of Pansomonadida, not Cercomonadida. In a thorough overhaul of glissomonad taxonomy we describe 29 new species, new genera Sandona, Neoheteromita, Flectomonas, Allapsa, and Teretomonas, and morphologically distinctive families: Sandonidae, Allapsidae, Bodomorphidae, and Proleptomonadidae.

Aurigamonas solis n. gen., n. sp., a soil-dwelling predator with unusual helioflagellate organisation and belonging to a novel clade within the Cercozoa.

A flagellate predator, Aurigamonas solis n. gen., n. sp., with numerous radiating axopodia-like appendages, has been isolated in culture from soils. Despite its heliozoan-like appearance, Aurigamonas is not a sit-and-wait predator but a mobile hunter and its stiff appendages are not microtubule-supported axopodia but elongate haptopodia, each supported by a cylindrical core of microfilaments and bearing at its capitate tip a single extrusome-like body (haptosome). Prey flagellates are trapped on the sticky tips of the haptopodia and a large funnel-shaped pseudopodium then emerges to engulf the prey or suck out part of it for internal digestion. Pseudopodial contact is accompanied by killing, possibly as a result of the injection of spicules by the predator. Cytoplasmic haptosomes appear to induce formation of a haptopodium on making contact with the plasma membrane. Propulsion of the organism along the substratum is effected by beating of a long trailing flagellum, the short and inconspicuous second flagellum lacks motility. Small subunit rDNA sequencing shows that Aurigamonas arose within the Cercozoa. Its closest relatives are Cercobodo agilis and several flagellates currently known only as environmental sequences. This conclusion is supported further by the presence of only a single amino acid insertion in the polyubiquitin sequence of Aurigamonas solis.

An updated view of kinetoplastid phylogeny using environmental sequences and a closer outgroup: proposal for a new classification of the class Kinetoplastea.

Given their ecological and medical importance, the classification of the kinetoplastid protists (class Kinetoplastea) has attracted much scientific attention for a long time. Morphology-based taxonomic schemes distinguished two major kinetoplastid groups: the strictly parasitic, uniflagellate trypanosomatids and the biflagellate bodonids. Molecular phylogenetic analyses based on 18S rRNA sequence comparison suggested that the trypanosomatids emerged from within the bodonids. However, these analyses revealed a huge evolutionary distance between the kinetoplastids and their closest relatives (euglenids and diplonemids) that makes very difficult the correct inference of the phylogenetic relationships between the different kinetoplastid groups. Using direct PCR amplification of 18S rRNA genes from hydrothermal vent samples, several new kinetoplastid-like sequences have been reported recently. Three of them emerge robustly at the base of the kinetoplastids, breaking the long branch leading to the euglenids and diplonemids. One of these sequences belongs to a close relative of Ichthyobodo necator (a fish parasite) and of the 'Perkinsiella amoebae'-like endosymbiont of Neoparamoeba spp. amoebae. The authors have studied the reliability of their basal position and used all these slow-evolving basal-emerging sequences as a close outgroup to analyse the phylogeny of the apical kinetoplastids. They thus find a much more stable and resolved kinetoplastid phylogeny, which supports the monophyly of groups that very often emerged as polyphyletic in the trees rooted using the traditional, distant outgroup sequences. A new classification of the class Kinetoplastea is proposed based on the results of the phylogenetic analysis presented. This class is now subdivided into two new subclasses, Prokinetoplastina (accommodating the basal species I. necator and 'Perkinsiella amoebae') and Metakinetoplastina (containing the Trypanosomatida together with three additional new orders: Eubodonida, Parabodonida and Neobodonida). The classification of the species formerly included in the genus Bodo is also revised, with the amendment of this genus and the genus Parabodo and the creation of a new genus, Neobodo.

Ribosomal RNA phylogeny of bodonid and diplonemid flagellates and the evolution of euglenozoa.

Euglenozoa is a major phylum of excavate protozoa (comprising euglenoids, kinetoplastids, and diplonemids) with highly unusual nuclear, mitochondrial, and chloroplast genomes. To improve understanding of euglenozoan evolution, we sequenced nuclear small-subunit rRNA genes from 34 bodonids (Bodo, Neobodo, Parabodo, Dimastigella-like, Rhynchobodo, Rhynchomonas, and unidentified strains), nine diplonemids (Diplonema, Rhynchopus), and a euglenoid (Entosiphon). Phylogenetic analysis reveals that diplonemids and bodonids are more diverse than previously recognised, but does not clearly establish the branching order of kinetoplastids, euglenoids, and diplonemids. Rhynchopus is holophyletic; parasitic species arose from within free-living species. Kinetoplastea (bodonids and trypanosomatids) are robustly holophyletic and comprise a major clade including all trypanosomatids and most bodonids ('core bodonids') and a very divergent minor one including Ichthyobodo. The root of the major kinetoplastid clade is probably between trypanosomatids and core bodonids. Core bodonids have three distinct subclades. Clade 1 has two distinct Rhynchobodo-like lineages; a lineage comprising Dimastigella and Rhynchomonas; and another including Cruzella and Neobodo. Clade 2 comprises Cryptobia/ Trypanoplasma, Procryptobia, and Parabodo. Clade 3 is an extensive Bodo saltans species complex. Neobodo designis is a vast genetically divergent species complex with mutually exclusive marine and freshwater subclades. Our analysis supports three phagotrophic euglenoid orders: Petalomonadida (holophyletic), Ploeotiida (probably holophyletic), Peranemida (paraphyletic).

The soil flagellate Proleptomonas faecicola: cell organisation and phylogeny suggest that the only described free-living trypanosomatid is not a kinetoplastid but has cercomonad affinities.

The only putative free-living trypanosomatid is Proleptomonas faecicola described first by Woodcock in 1916 as a coprophilic flagellate with striking Leptomonas-like flagellar movement but lacking a kinetoplast. P faecicola was later identified by Sandon in 1927 as a widespread non-phagotrophic inhabitant of soils. No division stages were seen by either observer. An organism conforming to Woodcock's light microscope description has been isolated from tapwater and cultivated axenically in various serum-containing media. Division has been shown to occur in an aflagellate stage enclosed in a thin cyst wall. Electron microscopy of the flagellate stage reveals that, in addition to the long locomotory flagellum, a second non-motile flagellum is present attached to the body along its entire length. The flagellate's ultrastructure lacks all the major features of the Trypanosomatidae. The several mitochondria of Proleptomonas have tubular cristae and lie between intracytoplasmic microtubules originating as a loose cone associated with the flagellar basal bodies. This cytoskeleton is much reduced in the division cyst. A comparable Proleptomonas-like flagellate with similar division cysts has been observed in soil samples from farmland. Phylogenetic analysis based on SSU rRNA gene sequences suggests that the cultured organism identified here as Proleptomonas is unrelated to the Kinetoplastida and has affinities with the Phylum Cercozoa Cavalier-Smith, even though in morphology, life cycle and mode of feeding it bears little resemblance to any member of that diverse grouping.