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http://www.elsevier.de/protisPublished online date 25 June 2009

e-mail david.b1Correspondin2Present addrMuseum, Cro3Joint first aut

& 2009 Elsevdoi:10.1016/j

160, 483—521, November 2009

Protist, Vol.

ORIGINAL PAPER

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

David Bassa,1,2,3, Alexis T. Howea,3, Alexandre P. Mylnikovb, Keith Vickermanc, Ema E. Chaoa,James Edwards Smallbonea, Jemma Snella, Charles Cabral Jra, and Thomas Cavalier-Smitha

aDepartment of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UKbInstitute for Biology of Inland Waters, Russian Academy of Sciences, Yaroslavskaya Oblast,Borok 152742, Russia

cDivision of Environmental and Evolutionary Biology, University of Glasgow, Glasgow G12 8QQ, UK

Submitted August 14, 2008; Accepted January 31, 2009Monitoring Editor: Michael Melkonian

Cercomonads (=Cercomonadida) are biflagellate gliding bacterivorous protozoa, abundant anddiverse in soil and freshwater. We establish 56 new species based on 165 cultures, differentialinterference contrast microscopy, and 18S and ITS2 rDNA sequencing, and a new genusCavernomonas studied by scanning electron microscopy. We fundamentally revise the phylogenyand classification of cercomonad Cercozoa. We describe 40 Cercomonas species (35 novel), sixEocercomonas (five novel), two Cavernomonas, and 18 Paracercomonas species (14 novel). Weobtained additional cercomonad clade A (Cercomonas, Eocercomonas, Cavernomonas) sequencesfrom multiple environmental DNA libraries. The most commonly cultivated genotypes are not thecommonest in environmental DNA, suggesting that cercomonad ecology is far more complex thanimplied 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 DNAlibraries regularly detect them in coastal marine sediments. The first ultrastructural study of ananaerobic cercozoan, Paracercomonas anaerobica sp. nov., a highly divergent cercomonad, showsmuch simpler ciliary roots than in clade A cercomonads, a ciliary hub-lattice and axosome, andmitochondria with tubular cristae, consistent with it being only facultatively anaerobic. We alsodescribe Agitata tremulans gen. et sp. nov., previously misidentified as Cercobodo (=Dimastiga-moeba) agilis Moroff.& 2009 Elsevier GmbH. All rights reserved.

Key words: Cercomonas; Eocercomonas; Paracercomonas; Cavernomonas; 18S rDNA phylogeny; Agitata.

ass@zoo.ox.ac.uk (D. Bass).g author; fax +44 1865 281310.

ess: Department of Zoology, The Natural Historymwell Road, London SW7 5BD, UKhors

ier GmbH. All rights reserved..protis.2009.01.004

Introduction

Cercomonads are cercozoan biflagellate hetero-trophs found globally in soil and freshwaterecosystems (Bass and Cavalier-Smith 2004; Basset al. 2007; Cavalier-Smith and Chao 2003;

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Ekelund et al. 2004). Like glissomonads, which wehave recently treated in similar detail (Howe et al.2009), they comprise one of the most commonand frequently encountered zooflagellate groupsin soil (Ekelund and Patterson 1997; Foissner1991; Sandon 1927), where they are thought tohave an important role in nutrient cycling (Mylni-kov and Karpov 2004). However, they are gen-erally much larger than glissomonads, with moremetabolic cells and greater intra-clonal variabilityboth in morphology and behaviour. Prior to thepresent study, at least 49 cercomonad specieswere named (Mylnikov and Karpov 2004), butmost have not been found since they were firstdescribed, DNA sequences and clonal culturesare available for very few, and some may be toopoorly described for accurate identification. Thetaxonomy of the group urgently requires revision(Al-Qassab et al. 2002; Foissner 1991). Our recentmolecular and morphological studies of clonalcultures suggested that there are many unde-scribed species and that some highly geneticallydivergent taxa are so similar morphologically as tobe readily confused (Karpov et al. 2006). Mole-cular phylogenies have shown that cercomonadsoccupy two very distinct clades (A and B) on 18SrDNA trees, which show a weak but consistentsister relationship in most well sampled distance,Bayesian, and maximum likelihood trees (Bassand Cavalier-Smith 2004; Bass et al. 2005;Cavalier-Smith and Chao 2003; Cavalier-Smithet al. 2008). Ultrastructural investigations of thekinetid structure of five strains from the bettersampled of these two clades (A) showed that theircytoskeletal features are complex and substan-tially different from those in all other protists(Karpov et al. 2006). Molecular phylogeneticanalyses in that study showed that clade Acomprises two subclades, which are very distinctboth ultrastructurally and genetically; on thesebases clade A was split into two genera: Cerco-monas and Eocercomonas. The morphologicallyand phylogenetically distinct clade B cercomo-nads were renamed Paracercomonas (Karpovet al. 2006).

The classical genus Cercomonas has beensubject to considerable change in taxonomy andnomenclature since Dujardin (1841) first estab-lished it. He described Cercomonas as havingcells rounded or discoid, tubercular, with ananterior flagellum and a variable posterior exten-sion, in the form of a tail that can be variable inlength and thickness. Later authors realized thatthe tail was a flagellum plus an associatedtapering extension. Dujardin gave rather vague

and generalised descriptions of eleven species, ofwhich five are still considered to be cercomonads:Cercomonas crassicauda, C. cylindrica, C. fusi-formis, C. globosus, and C. longicauda. His otherspecies, C. acuminata and C. detracta, C.lacryma, C. lobata, C. truncata, and C. viridis,were thought by Kent (1882) to be transitionalstages of other species; they may simply be yetuncharacterized free-living zooflagellates. Someindividual cercomonad species have convolutedtaxonomic histories. A typical example is Cerco-monas longicauda, first described by Dujardin(1841) as having a fusiform and flexible body,8-9mm in length; the posterior end of the cellterminating in a long, thin, flexible filamentextending approximately 15mm; the anterior fla-gellum was between 30 and 40mm. Stein (1878)re-described this species as an organism with abody length of 23-36mm (four times that ofDujardin), an anterior flagellum only one and ahalf times the body length, and a posteriorflagellum twice the body-length. Kent (1882) thendescribed a 9.5mm long C. longicauda with aposterior flagellum twice the length of the body,and an anterior flagellum shorter than the poster-ior. Klebs (1892) described ‘Dimorpha’ longicauda(Dujardin) as 18-36mm long and having a posterior‘tail’ that distinguished this species from others bybeing sometimes single, sometimes branched orribbon-like; the cell was also said to shownumerous branching pseudopodia. Lemmerman(1913) noted the difference between the originaldescription by Dujardin (1841) and Stein (1878),and described ‘Cercobodo’ longicauda (Stein) asa somewhat metabolic oval to spindleform cell,18-36 mm long, with a tail-like posterior end,flagella of equal length, both around body length;simple or branching pseudopodia. Skuja (1939)then described ‘Cercobodo’ longicauda (Stein) asa 20-49 mm long oval, distorted oval, or club-shaped cell; with or without a sometimes bifurcat-ing long posterior tail; an anterior flagellum ofapproximately body length, and a posteriorflagellum half to three quarters body length; finesingle or branching ‘rhizopodien’. More recentdescriptions are closer to Cercomonas longicauda(Dujardin 1841) in size (Ekelund et al. 2004;Zhukov 1971), although none noted an anteriorflagellum three to four times body length, includingthe Cercomonas longicauda Dujardin neotypedesignated by Karpov et al. (2006).

Similar problems exist in other species, high-lighting two important sources of confusion incercomonad taxonomy: the tendency of authorsto associate different generic names with the

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group; and the lack of precision in descriptions ofeach strain, either through error or an assumptionthat the basic cercomonad morphology repre-sented only a relatively few, easily distinguishabletypes. These sources of error are fed andcompounded by two more aspects of cercomo-nad biology: 1) the highly variable morphology ofthe cells on the scales of seconds and minutes(flexible cells with frequent and varied pseudopo-dial tendencies that can differ greatly in morphol-ogy and size when feeding, moving, or interactingwith other individuals), and days and weeks(switches between highly motile flagellate andmore stationary amoeboid stages, formation ofcysts and other resting states, and various cellaggregations and plasmodial structures); 2) thevery high genetic diversity within the group (Bassand Cavalier-Smith 2004; Bass et al. 2007; Karpovet al. 2006), which is far greater than the number ofeasily accessible morphological characters pre-viously considered sufficient to distinguishbetween them. Thus two independent isolates,even if superficially similar, are less likely torepresent the same genotype than previouslyassumed.

To put cercomonad taxonomy on a sounderbasis we have isolated well over a hundred newclonal cercomonad strains and studied them andstrains from available collections by differentialinterference contrast (DIC) high definition videomicroscopy and 18S rRNA gene sequencing andphylogeny. We have been able to assign only 10existing species names to our 165 cultures, whichemphasises how few species have yet been welldescribed, and have established 56 new speciesfor many of the others. In each case, we havedescribed as many of the variable characters foreach strain as possible, and taken photographsand videos at a consistent and easily attainablegrowth phase. The 18S rDNA and ITS2 sequencesprovide a definitive marker of identity. Two newspecies were also studied by electron micro-scopy: scanning electron microscopy for the typespecies of the new clade A genus, Cavernomonas,which is much more rigid than previously knowngenera, and thin sectioning for the first describedanaerobic cercozoan: Paracercomonas anaero-bica. In addition to our extensive new observa-tions we have sought to maximize continuity withthe past by including the results of a comprehen-sive literature survey of cercomonads, to reduceconfusion about their past classification andnomenclature.

Our critical evaluation of the literature andcombined morphological and molecular studies

of many clonal cultures should provide a solidbasis for future studies. We conclude that theremust be at least hundreds of cercomonad speciesand discuss the ecological and phylogeneticsignificance of our results. Since many Cercomo-nadidae have in the past been confused withCercobodo and all regarded as congeneric wealso studied microscopically a culture (CCAP1901/1) that was named Cercobodo agilis, butwhich phylogenetically does not group withCercomonadidae, but with Aurigomonas - as thenew order Pansomonadida (Vickerman et al.2005). We show that CCAP 1901/1 was misidenti-fied and is a new species and genus: Agitatatremulans. In a correspondingly detailed study ofHeteromitidae (Howe et al. 2009), formerly alsoplaced in Cercomonadida, we concluded that thefamily was misnamed and is also probably not asister to Cercomonadidae, but to Pansomona-dida. Therefore, ‘heteromitids’ were excludedfrom Cercomonadida as a novel order Glissomo-nadida (Howe et al. 2009). Thus Cercomonadidanow includes only the family Cercomonadidae(clades A and B); as before (Karpov et al. 2006) thevernacular term ‘cercomonad’ refers explicitlyonly to Cercomonadidae (i.e. all Cercomonadidain this new stricter sense), embracing only the fourgenera recognised here.

Results

The extensive results are presented in six parts: 1)Diagnoses of the 56 new cultured species as seenby DIC microscopy, plus 10 others for which weadopt a previously published name (with neotypi-fications where necessary) (Figs 1—11), and 15further strains for which we have sequence databut no surviving culture and therefore too poorquality images/videos to justify describing them asnew species in this paper. Our designation of typematerials is explained below. For some speciesthere is a ‘further observations’ entry in thedescriptions; these are generally strains observedrepeatedly under different conditions over severalmonths, for which we can therefore provide moreextensive information than for others. Obviously wedo not know how other species would haveperformed under these conditions. 2) Electronmicroscopy of Paracercomonas anaerobica(Fig. 12). 3) A taxonomic and nomenclaturalhistory of cercomonads - a literature surveyreport and rationalisation of previous names forcercomonads - and assessment of historicalsynonyms. 4) Summary of nomenclatural

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changes. 5) Current status of the genusCercobodo and diagnosis of Agitata tremulans n.gen., n. sp. (Fig. 13). 6) 18S rRNA phylogenetictrees showing the relationships among the variouscercomonad groups and their relationship toAgitata and other Cercozoa (Figs 14 and 15).

We also constructed cercomonad clade A-specific (but see Discussion) environmental 18SrDNA libraries from numerous habitats in manycountries for sequencing. Only environmentalsequences thus found that were identical to thosefrom cultured strains are mentioned in Supple-mentary Table S1, which summarises the prove-nances of cultures and environmental sequencesfor each species and the frequency with whicheach was found. Some cultures died after weobtained their sequences but before we couldstudy them thoroughly enough microscopically toidentify them unambiguously; such morphologicalobservations as we have for them are briefly notedas a basis for further study.

Diagnoses and Descriptions of AllStrains

Type Materials

Permanent preparations or fixed cultures may beuseful for protozoa with features that are wellpreserved by such techniques (e.g. ciliate pelli-cles; silica scales and tests), but for small soft-bodied flagellates like cercomonads they wouldbe taxonomically useless, leading to loss ofdelicate structures like filopodia, distorting lengthrelationships between cell bodies and flagella, andfailing to capture the most reliable characters fordiscriminating between strains. We have done ourbest to follow the International Code for ZoologicalNomenclature (ICZN), but stress that the latestrevision is inadequate for such protozoa andneeds urgent revision. It is apparently contra-dictory - in places in the absence of permanentspecimens it appears to require a type cultureinstead, but in others it appears to allow a typeillustration instead; we interpret it as allowingeither, as in previous versions. Nowhere does itprovide for (nor disallow) a type DNA sequence,which in many protozoa is more reliable thanmorphology for identification. Reliance on a protistculture alone as type is dangerous. Even in thebest collections strains may die, become over-grown by a previously low-level contaminant, orbe mis-labelled during subculturing. It is thereforemost satisfactory to have a culture, illustration,

and a discriminating type sequence for those newprotozoan species for which preserved specimensare virtually useless. The corruption of any one ofthese, by mis-labelling, death, or contaminationwill then be easier to detect. Continuing concor-dance between the three kinds of data will supporttheir veracity; if they become discordant sometime in the future, existing ICZN provisions allowambiguities to be resolved.

Pseudopodia

Different pseudopodial forms are one of the moststriking aspects of cercomonad morphology.Seven distinct types have been observed. To aidconsistent interpretation of our descriptions,Figure 1 shows the five commonest. Two othersare rarely seen: axopodia (as in C. radiata, Fig. 8A)and filose reticulate (i.e. anastomosing) pseudo-podia (as in E. ramosa, Fig. 8G).

Flagellar Proportions

Flagella lengths are expressed as multiples of celllength, based as far as possible on cells moving inone direction, not on amoeboid or distorted forms.This appears to be a more reliable indicator ofrelative flagellar length than simple measurementsas the ratio of flagellar to body lengths remainsquite constant for differently sized cells of thesame strain; there must therefore generally be aproportional length control.

Cysts and Cercomonad Life Histories

Most cultures were examined for cysts. Ourobservations are reported within the species diag-noses, below. In general it is difficult or impossible todistinguish between cysts of different species underthe light microscope as they mostly lack distinctivecharacters and often vary significantly in size withina clonal culture, and to a lesser extent in shape andsurface texture. Therefore, the size ranges givenneed not be definitive. Under our standard cultureconditions cercomonad strains differed in theirpropensity to form cysts, some cultures becomingdensely packed with them, others with very few.This was usually consistent across replicate culturesof the same strain under identical conditions. Also,the interval after inoculation and density of activecells at which cysts begin to form differs amongstrains, perhaps reflecting inter-strain differences intolerance to our standard culture conditions. How-ever, it is impossible to say that a culture neverforms cysts; in several cases we saw cysts (if only

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Figure 1. Five commonly seen types of cercomonad pseudopodia. Micrographs not to scale. a Lamellar bBulbous c Filose-branching d Filose, and e Finger-like. Not shown are the rarer filose reticulate pseudopodia(where filose pseudopodia anastomose, as seen in E. ramosa strain C-80 Fig. 8G) and axopodia (as seen inC. radiata Fig. 8A).

487Cercomonad Phylogeny and Classification

one or two) in cultures of strains thought not to formcysts even after frequent observations over manyyears. Conditions under which cysts are formedmay vary widely among strains.

Such differences may represent variation in lifehistory strategies and r/K-selection, and may beas pronounced even under optimal conditions foreach strain. Differences in life history strategiesmay provide more informative characters fordistinguishing cercomonad species; our observa-tions point to this being an important area ofinvestigation if their biology is to be more fullyunderstood.

Cyst measurements are from replicate observa-tions per strain; they are mostly not wholenumbers because they have been scaled to adjustfor the magnification of the images measured.Cyst descriptions are given in the following format:range of diameters observed, regularity of circularoutline, texture of cyst wall, average density incultures. Note that in some Paracercomonas

species it can be difficult to distinguish betweencysts and very inactive, spherical cells withindistinct or withdrawn flagella.

The vast majority of the light micrographs in thispaper were taken at a fixed interval of 24 hoursafter inoculation to optimize optical quality andprovide procedural consistency. This tends toselect for cells in their actively gliding motilephase, which makes description easier, butobviously reduces the observer’s exposure tomore amoeboid and resting stages. Cells in thesestages are often stationary, potentially exhibitingthe full range of pseudopodial extension andmovement characteristic of that species, some-times accompanied by high levels of cytoplasmicmovement. This is often, but apparently notalways, associated with food (bacteria) capture.Non-encysted resting stages are usually roundedcells, sometimes with a flagellum extended quitestraight and motionless, or gently moving. Ageneral observation is that from c. 4 days after

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inoculation, when bacterial densities increase,cercomonad cells become slower and less directgliders, more amoeboid, and with a wider range ofcell morphologies and activities, as interactionsbetween themselves and their prey become moreconcentrated and complex. Another generalobservation is that apparently very few cercomo-nads are capable of swimming, free of thesubstrate, and maintaining directional locomotionin water. Only three species were seen to do this:Cercomonas karpovi (isolated from a river: mostspecies were from soil), Paracercomonas meta-bolica, and P. saepenatans. However, more inten-sive observation might show other speciescapable of this, even if only rarely or under veryspecific conditions.

Where we have information about strains inolder cultures it is provided in the descriptions anddiagnoses below, but it is not available for allstrains and later observations are not at set pointsin time. To undertake such extended observationsof all strains in a structured way would have beenimpossible in a study of this scale. Some strainshave been thus studied, showing that closelyrelated and morphologically similar strains canvary consistently in the timing and nature of lifehistory stages in replicated laboratory cultures,and that single strains can show a wide range ofmorphologies under a range of different cultureconditions (C. Cabral, J. Snell, J. Edwards-Smallbone, unpublished data). Some of thesedata are summarized below, highlighting thepotential of culture conditions to confuse attemptsto describe and identify cercomonad strains in thelaboratory.

Culture Conditions

Three strains - C-43 (Cercomonas dactyloptera),17-12D (C. clavideferens), and C-71 (Paracerco-monas virgaria) - were studied in detail to evaluateeffects of different media on cell characteristicsand behaviour. The effects of different artificialseawater (ASW) concentrations were complex. At1% ASW cells all three strains became signifi-cantly larger than the freshwater control. At 10%and 25% ASW cells of C-43 and C-71 becamesmaller, but 17-12D failed to grow and theinoculated cells died. Four different non-salinemedia were also tested-two inorganic (Prescott’sand James’s; Chalkley’s) and two with multipleorganic supplements - Modified Chang’s Serum-Casein-Glucose-Yeast Extract; Jones’s HorseSerum). The different media had significant effectson cell size for all strains, but not in a consistent

direction among strains. 17-12D was intolerant ofswitches in media and frequently died. There wasno consistent difference between responses toorganic or inorganic media either in the directionor degree of response. Cysts did not develop inthe organic media, and were often smaller in ASW.Type of medium also influenced the prevalenceand nature of plasmodia. Cell morphology andsize changed significantly with time in individual(replicated) cultures for C-43 but not for C-71.These findings emphasise that reliance on mor-phological characters for defining species in suchvariable organisms is fraught with danger.Although we have described each strain as fullyas possible, sequence data must be consideredalongside morphology for reliable species identi-fication - and future diagnoses of cercomonadspecies.

Species Boundaries

Species boundaries were decided on the bases ofrDNA sequence identity and phenotypic charac-ters observed by light microscopy of at least oneclonal culture of each strain (more whereveravailable). Because of the high morphologicaland behavioural plasticity of many/most cerco-monad strains, uniqueness of an appropriategenetic marker is the most reliable way ofdistinguishing between species. Such sequencesenable unambiguous digital identification, inde-pendent of culture conditions and intensity ofobservational effort, and are more future-proofthan relying on a graded phenotypic characterwhich might subsequently be observed in otherspecies listed here (or in still unknown strains) thatotherwise deserve to be separate species. There-fore, in all diagnoses below, whether or not aparticular phenotypic character apparently distin-guishes a species from all others, rDNA identity isthe overriding character used to define speciesand separate them from others. Specifically, wecompared the most variable regions of thecercomonad 18S rDNA - V2, V4, V5, and V7(Wuyts et al. 2000) among strains (as described inMethods), to reduce artefactual effects of sequen-cing errors and intra-clonal polymorphism alongthe whole gene. Those that differed unambigu-ously in 18S or in more than one helix of ITS2rDNA were regarded as separate species. Wejustify splitting species at this level of phylogeneticresolution in two ways: 1) in a recent study wefound that strains with identical 18S rDNAbut different ITS rDNA differed significantlyin behaviour and/or physiology in controlled

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experiments to require separate consideration,and 2) particular ITS2 rDNA sequence differencesamong strains are increasingly recognised as abroadly applicable marker of boundaries betweensexual species (Coleman 2007; Muller et al. 2007).ITS rDNA can provide a sensible marker fordefining species (or at least OTUs/surrogatespecies in putative asexuals), and for use inecological studies, as exemplified for cryptomo-nads (Hoef-Emden 2007; Hoef-Emden and Melk-onian 2003). Therefore strains differing in 18Ssequence are very likely to represent clearlydistinct species. Because in some cases thedistinction between 18S sequences was ambig-uous we sequenced the ITS2 of as many strainsas possible to verify their distinctiveness. The ITS2sequences ranged from 230 to 400 bp in length,with no significant differences between genera.There are three or four helices, the fourth oftenvery small or absent. In general ITS2 sequenceswere unambiguous, and identical reads wereobtained for independent isolates of many spe-cies. However, for some members of clade A1band Group B1b and a relatively divergent sub-clade of A1a the ITS2 did not sequence easily,either consistently failing or producing mixedtraces on multiple independent attempts (startingfrom separate DNA extractions). Therefore foreach species we designate the type sequenceas the combined V2+V4+V5+V7 18S rDNAsequence, in combination with ITS2 where avail-able. Most of our new species are clearly distinctat the 18S level; generally the most closely relatednew species differ unambiguously in the stemregions of at least two of ITS2 helices 1-3. We donot know whether cercomonads are sexual, so thecompensatory base changes (CBCs) coincidingwith biological species boundaries identified byColeman (2007) do not apply here. However, in thevast majority of cases levels of ITS2 divergenceeven between our most closely related speciesapproximates to that between known sexualspecies; as this also coincides with clearlydifferent cercomonad strain characters, it is themost appropriate basis for separating speciescurrently available.

Conventions and Abbreviations

In view of the large number of new namedspecies, to make treatments of each more concisewe do not repeat the following conventions andtaxonomic policy that apply to them all. Where oursequences match ones in the literature, referencesare given. For each new species or neotypes of

old ones, the isolator, type locality, and date aregiven in parentheses after the strain name withoutrepeating the word ‘type’. Then illustrations oftype material are indicated, and the GenBankaccession number of the type strain is given.Abbreviations: CV=contractile vacuole(s); N=nucleus; AF=anterior flagellum; PF=posterior fla-gellum; BL=body length. ‘Metabolic’ is a compo-site measure of the degree to which the cellchanges shape, and how rapid shape changesare. No directional, locomotive movement isimplied by this term. A cell need not formpseudopodia to be highly metabolic. Oxfordmeans Oxford, UK. Left and right refer to thecell’s left or right, i.e. as seen from above by adipping objective; dorsal (upper surface) andventral (lower surface, attached to substratum)are also from this perspective. Coll.=collector.Strains are listed in the order in which they occuron Figure 14, so that most closely related strainscan be easily compared.

Family Cercomonadidae Kent 1880 sensuKarpov et al. (2006)

Additional information: contrary to what we pre-viously thought (Karpov et al. 2006), the posteriorflagellum of all cercomonads studied - exceptCavernomonas - closely associates with the fulllength of the cell for the majority of the time, incontrast to Agitata tremulans n. sp. and glisso-monads; its occasional separation in some spe-cies of Cercomonas, Eocercomonas, and Para-cercomonas is transient.

Genus Cercomonas Dujardin 1841 emend. Karpovet al. 2006 (Figs 2—7)

Diagnosis in Karpov et al. (2006). Type species C. longicaudaDujardin, 1841 (neotypified: Karpov et al. (2006); neotypeCCAP 1910/2).

Variably sized gliding biflagellates, often with long flagella,and including the largest known cercomonads; mostlyrounded or finger-like pseudopodia. We silently corrected tothe feminine specific names of four species originallyCercobodo but transferred to Cercomonas by Mylnikov andKarpov (2004) with incorrect masculine spelling.

Cercomonas media Bass and Cavalier-Smith sp. nov.Figure 3A. Type strain: xt182 (DB; Oxford; 2006). Diagnosis:Length: 8-14mm. AF 1.5-2X BL; PF 1.5-2X BL. AF movement alicking motion, most active at distal tip; occasionally heldextended with little movement. Cell movement direct andquite rapid. Occasional lamellar, finger-like, or filose cytoplas-mic tail. Lamellar, finger-like and filose pseudopodia onposterior of cell. Not very metabolic. Laterally positionedCVs in posterior half of cell. Cysts 8.0-8.5 mm, slightly irregular,smooth, sparse. 18S-type isolated 7 times: 3 in 2006 from

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Figure 2. Cercomonas clade A1a1. The scale is constant for all figures; names and cell length range aregiven for each. A: C. phylloplana B: C. nebulosa C: C. lenta D: C. mutans E: C. volcana.

490 D. Bass et al.

winter grassland soil near Oxford (AH); twice from pondsediment, Priest Pot, Cumbria, UK, once from New Zealandsoil, once from USA soil (all DB). Type sequence: 18S: FJ790681.

Etym. medius L. medium, ordinary.Cercomonas longicauda Dujardin, 1841 Figure 3B. Neo-

type strain: CCAP 1910/2 (see Karpov et al. 2006). Descrip-tion: Length: 8-14 mm. AF 1.5-2X BL; PF 1.5-2.25X BL. Cellmovement very slow, indirect. Long, filose cytoplasmic tail,can become lamellar. Fan-shape and finger-like pseudopodiaall over cell. Very metabolic. Several CVs in various locations,but often lateral on periphery. Cysts 4-8mm, regular to slightlyirregular/ovoid, smooth, sometimes dense. Type sequence:18S: DQ442884; ITS2: FJ797436. Further observations:forms plasmodia. Cells can become rounded, inactive, andwith extended AF. 18S-type isolated 4 times in Europe: first in2001 from freshwater, England, UK (M. Zolffel); in 2001 fromlake water, Plon, Germany (APM); from soil, Holland; oncefrom UK soil by J. Darbyshire.

Sequences generated in this study confirm that the C.longicauda 18S-type is also shared by Cercomonas sp. 21ATCC50317 (GenBank 18S: FJ790680) and is extremelysimilar to that of Cercomonas sp. 22 ATCC50318 (GenBank18S: FJ790679), contrary to the situation suggested by older,poorer quality sequences analysed in Cavalier-Smith andChao (2003) and Karpov et al. (2006). ITS2 sequences of thesetwo strains differ from each other and from CCAP 1910/2.Therefore, we designate both strains Cercomonas aff. long-icauda pending further studies.

Cercomonas hederae Howe and Cavalier-Smith sp. nov.Figure 3D. Type strain: IVY20 (AH; Oxford; 2006); NIES-2439.Diagnosis: Length: 10-18 mm. AF 1.5-2X BL; PF 1.5-2X BL. AFmovement relatively slow probing and flickering; occasionallymovement restricted to distal half. Cell movement quite slow,changes in direction frequent; occasional spurts of more rapidand direct movement. Filose, lamellar and finger-like cytoplas-mic tails. Bulbous pseudopodia especially at anterior andposterior; lamellar pseudopodia more often lateral. Severalfinger-like and filose pseudopodia often present at posterior.Pseudopodia granular. Moderately metabolic. 1 CV at posteriorend. Cysts 5.5-8.2 mm, regular to slightly irregular, smooth,medium-dense. 18S-type isolated three times in 2006 fromdifferent ivy leaves, Oxford, UK (AH). Type sequence: 18S:FJ790682; ITS2: FJ797437.

Etym. hederae L. of ivy.Cercomonas zhukovi Bass and Cavalier-Smith sp. nov.

Type strain: xt202 (DB; Borok, Russia; 2003). Diagnosis:morphologically indistinguishable from C. mtoleri, but ITS2rDNA sequence clearly different in helices 1 and 3. Isolatedonce from pond water. Type sequence: 18S: FJ790683; ITS2:FJ797438.

Etym. after BF Zhukov, Russian contributor to under-standing zooflagellate diversity.

Cercomonas mtoleri Howe and Cavalier-Smith sp. nov.Figure 3E. Type strain: BuffaloH5 (AH; Arusha NP, Tanzania;2007). Diagnosis: Length: 8-11 mm. AF 1.5-2X BL; PF 1.5-2XBL. AF movement a rapid flickering. Cell movement medium

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Figure 3. Cercomonas clade A1a1 continued. The scale is constant for all figures; names and cell lengthrange are given for each. A: C. media B: C. longicauda C: C. wylezichi D: C. hederae E: C. mtoleri F: C.hiberna G: C. deformans.

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to slow, often remaining in one location, flickering and probingwith AF. Filose, lamellar and finger-like cytoplasmic tail.Lamellar and finger-like pseudopodia all around cell exceptanterior; bulbous pseudopodia in posterior. Very metabolic. 1CV in posterior tip of cell, occasionally a second found in

posterior half. Cysts 6.4-9.1 mm, regular to irregular, smooth,dense. Isolated once; from buffalo dung. Type sequence: 18S:FJ790684; ITS2: FJ797439.

Etym. after William Mtolera, who helped DB collect samplesfrom Tanzania.

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Cercomonas hiberna Howe and Cavalier-Smith sp. nov.Figure 3F. Type strain: W70 (AH; Oxford; 2006); NIES-2440.Diagnosis: Length: 7-13 mm. AF 2-2.5X BL; PF 1.75-2.25X BL.AF movement slow gentle sweeps. Cell movement slow, oftenremain in one location; travel very little. Lamellar, finger-likeand filose cytoplasmic tail. Lamellar and finger-like pseudo-podia all over cell, but predominantly on left side. Occasionalfilose pseudopodia. Very metabolic. 1-2 CVs: usually 1 onright side of cell, the other in posterior half. Cysts 4.5-8.0 mm,regular, knobbled, medium-dense. 18S-type isolated twicein 2006 from two different winter grassland soil samples,near Oxford (AH). Type sequence: 18S: FJ790685; ITS2:FJ797440.

Etym. hibernus L. wintry, of/for winter time when it wasisolated.

Cercomonas wylezichi Bass and Cavalier-Smith sp. nov.Figure 3C. Type strain: HFCC89 (=Cercomonas sp.; Wylezichet al. 2007, Lake Schoehsee, near Plon, Germany). Diag-nosis: Length 5-10 mm. AF 1.5-2X BL; PF 1.5-2X BL. AFmovement occasionally very rapid flickering, though proximalquarter often remains still. Cell movement often direct, atmoderate speed, cells flexible, curve during changes indirection. Rare cytoplasmic tail. Bulbous, finger-like, filoseand lamellar pseudopodia. Not very metabolic. 1 CV usually inposterior half or central. Cysts 2.7-4.0 mm, regular to slightlyirregular/ovoid, smooth, very sparse. Type sequence: 18S:DQ211598; ITS2: FJ854695.

Etym. After C. Wylezich, the culture isolator.Cercomonas deformans Bass and Cavalier-Smith sp. nov.

Figure 3G. Type strain: 20-8D (DB; Oxford; 2003). Diagnosis:Length: 10-17 mm. AF 2-3X BL; PF 2X BL. AF movementflickering and sweeping, slow to rapid. Cell movement indirectand relatively rapid, or remain in one location and pivot.Finger-like and filose cytoplasmic tail. Pseudopodia form fromall over cell: most often very thin, extensive lamellarpseudopodia; less often finger-like and filose. Extremelymetabolic. 1 central CV, several more in posterior part. Formplasmodia. Cysts: no data. Isolated 4 times: once fromOxford; twice from wet rainforest soil, Panama, and oncefrom Malaysia soil (coll. 2002, TCS) (all DB). Type sequence:18S: FJ790686; ITS2: FJ797441.

Etym. deformare L. to transform, disfigure.Cercomonas ricae Howe and Cavalier-Smith sp. nov.

Figure 4A. Type strain: IB3 (AH; Brazil; 2006). Diagnosis:Length: 10-15 mm. AF 1.5-2X BL; PF 1-1.5X BL. Movement ofAF mostly in distal tip, often rapid ‘casting-out’ motion. Cellmovement occasionally rapid. Usually very long and filosecytoplasmic tail present; occasionally finger-like or lamellar.Lamellar, filose and finger-like pseudopodia all around cell.Extremely metabolic. 2 CVs, close to each other at mid toposterior end. Cysts �8mm (few seen), regular to slightlyirregular, smooth. Isolated once from lateritic soil from Brazil(coll. 2006 TCS). Type sequence: 18S: FJ790687; ITS2:FJ797442.

Etym. rica L. veil, cloak.Cercomonas sp. Strain Panama69 Notes: Length: 7.5-

11.5 mm. AF c. 2.5X BL; PF c.2X BL. AF movement usuallyquite slow and flickering; occasionally held extended withmovement restricted to tip.

Cell movement quite slow; posterior pseudopodiaextended and retracted, giving a ‘walking’ effect. Cytoplasmictail unknown. Quite metabolic. Cysts: no data. 18S-type (18S:FJ790688) isolated twice in 2003 from rainforest soil, Panama(DB), differing in ITS2 sequence.

Cercomonas paravarians Bass and Cavalier-Smith sp. nov. Figure 4B. Type strain: NZ1-7E (Sophie von

der Heyden; New Zealand; 2003). Diagnosis: Length: 11-17 mm. AF 1.5-2X BL; PF 1.3X BL. AF movement liquid; heldrelatively still with movement restricted to tip when progres-sing. Cell progresses with medium speed. Frequent lamellarand finger-like cytoplasmic tail. Lamellar pseudopodia atposterior end, more often on left than right. Metabolic. 1 CV inposterior on left. Cysts 3.6-7.3 mm, regular to slightly irregular,smooth to knobbled, dense. Type sequence: 18S: AY884322;ITS2: FJ797443. Further observations: cells can formclusters in which they sometimes become more spherical,and can form true holoplasmodia. Cysts may also occur indense clusters. 18S-type isolated 7 times: twice in 2006 fromsummer grassland soil near Oxford, once from moist sand in astreambed, central Chile (coll. TCS), all by AH; 3 times fromsoil from Oxford, once from soil from Borok, Russia, once fromsoil, New Zealand, all by DB.

Etym. varians from Cercomonas (=Cercobodo) varians(Skuja 1948), a very similar species though described ashaving 2 anterior CVs, unlike our isolate.

Strain AND25 of Lara et al. (2007) has the same 18S-type,but its ITS rDNA sequence is unknown.

Cercomonas diparavarians Bass and Cavalier-Smith sp.nov. Type strain: SA-L (ATCC PRA-21; Cavalier-Smith andChao (2003)) (E. Chao; garden soil, Cape Town,South Africa; 2000). Diagnosis: morphologically indistinguish-able from C. paravarians, but ITS2 rDNA sequence stronglydiffers in helices 1, 2, and 3. Type sequence: 18S: AF411266;ITS2: FJ797444.

Etym. paravarians; from C. paravarians, a closely relatedand similar species.

Cercomonas phylloplana Howe and Cavalier-Smith sp. nov. Figure 2A. Type strain: IIgrassA (AH; Oxford;2006); CCAP1910/6. Diagnosis: Length: 7-10 mm. AF 0.75-1XBL; PF 1.25-1.5X BL. AF proximal quarter remains relativelystill, distal end often relaxed and waving. Cell moves quitefast and directly. Occasional filose cytoplasmic tail. Bulbous(B), finger-like (E), and lamellar (A) pseudopodia onposterior half of cell, and both sides. Not very metabolic.1 mid to posterior CV. Cysts 7.3-8.2 mm, regular toslightly irregular, smooth, sparse. Isolated once from ablade of grass. Type sequence: 18S: FJ790689; ITS2:FJ797446.

Etym. phylloplane from where isolated, the surface of a leaf.Cercomonas nebulosa Bass and Cavalier-

Smith sp. nov. Figure 2B. Type strain: xt164 (EC; Khao YaiNational Park, Thailand; 2001). Diagnosis: Length: 7-12 mm.AF 1.5-2X BL; PF 1.5-2X BL, tapered. AF repeatedly with-drawn and re-extended, or extended forward probing up intomedium, left and right. Cell movement slow and indirect;frequently pauses to pivot, though usually progressing.Cytoplasmic tail can be very long and thin, and ‘beaded’; orfinger-like as part of cell’s general metabolism. Fan-shaped,finger-like, filose (D), and bulbous pseudopodia form all overcell. Extremely metabolic. 1-3 CVs in various locations, and ofvarious sizes. Cysts 6.4-6.8 mm, slightly irregular, smooth toknobbled surface, sparse. Isolated once from rainforest soilnear flooded stream in Khao Yai National Park, Thailand,collected by TCS, later cultured by EC. Type sequence: 18S:FJ790690.

Etym. nebulosus L. cloudy, because of irregular shape.Cercomonas lenta Howe and Cavalier-Smith sp. nov.

Figure 2C. Type strain: WA84 (AH; Oxford; 2006). Diagnosis:Length: 10-21 mm. AF 1.5X BL; PF1.5-2X BL. AF movementliquid. Cell movement slow. Occasional cytoplasmic tail,finger-like or filose, more often on left. Lamellar pseudopodiaall over cell, but especially on left; bulbous pseudopodia

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Figure 4. Cercomonas clade A1a1 continued. The scale is constant for all figures; names and cell lengthrange are given for each. A: C. ricae B: C. paravarians C: C. laeva D: C. kiaerdammane E: C. rapida F: C.ambigua G: C. parambigua H. C. paraglobosa.

493Cercomonad Phylogeny and Classification

usually on anterior. Filose pseudopodia occasional. Verymetabolic. 1-3 CVs in various positions, usually at posteriorend. Cysts 6.4-8.2mm, irregular, knobbled, very sparse, oftenclustered. Isolated once; grassland soil near Oxford. Typesequence: 18S: FJ790691; ITS2: FJ797447.

Etym. lentus L. Slow, sluggish.

Cercomonas mutans Howe and Cavalier-Smith sp. nov. Figure 2D. Type strain: W40a (AH; Oxford;2006); NIES-2444. Diagnosis: Length: 6-10 mm. AF 2X BL; PF2X BL. AF movement quite slow, with fluid waving motion. Cellmovement slow; frequent directional changes. Occasionallamellar cytoplasmic tail does not persist. Lamellar pseudopodia

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on posterior, left and right. Very metabolic. 1-2 CVs laterallypositioned. Cysts 2.7-5.0 mm, regular to slightly irregular,smooth, medium-dense. Isolated once; grassland soil nearOxford. Type sequence: 18S: FJ790692.

Etym. mutare L. to change, alter, because of highly variableshape.

Cercomonas volcana Howe and Cavalier-Smith sp. nov. Figure 2E. Type strain: C18 (AH; Chile; 2006).Diagnosis: Length: 6-10 mm. AF 1.75-2.25X BL; PF 1.75-2.25X BL. AF movement slow, waving along its full length. Cellmovement slow, often stationary, probing in various direc-tions. Cytoplasmic tail short, brief, filose. Lamellar pseudo-podia on left and right side, most often at posterior end. Cellsomewhat metabolic. Some very large vacuoles. Cysts 3.6-5.5 mm, regular, smooth, sparse. Isolated once from moss-covered volcanic soil, central Chile (coll. TCS). Typesequence: 18S: FJ790693; ITS2: FJ797448.

Etym. volcanic from the volcanic soil type, and its vacuoles’crater-like appearance.

Cercomonas rapida Bass and Cavalier-Smith sp. nov.Figure 4E. Type strain: 18-6E (DB; Oxford; 2003). Diagnosis:Length: 11-15mm AF 1.25-1.5X BL; PF 1.25-1.5X BL. AFmovement quite a slow, smooth, wave-like whipping motion,covering a small arc ahead of moving cell. Cell movementrelatively rapid and direct, pauses frequent, changes indirection less so. Fan-shaped and finger-like pseudopodiaall over cell, more frequently on left. Very metabolic. 2 CVs, onleft and right sides. Cysts not seen. 18S-type isolated twice:once from freshwater from Oxfordshire, and once from soilfrom Russia. Type sequence: 18S: FJ790694; ITS2: FJ797452.

Etym. rapidus L. swift, rapid.Cercomonas laeva Bass, Mylnikov and Cavalier-Smith sp.

nov. Figure 4C. Phase contrast micrographs in Karpov et al.2006. Type strain: C-72 (A.P. Mylnikov; Borok, Russia; 2000);NIES-2441 (=C-56, Mylnikov (unpublished)). Diagnosis:Length: 6-12mm. AF 1.3-1.5X BL; PF 1.3-1.5X BL, acrone-matic. AF movement smooth and sweeping. Cell movementslow and indirect, though usually progressing. Long, filosecytoplasmic tail very often present. Lamellar, finger-like, filose,and filose-branching (C) pseudopodia all over cell, most oftenon left side. Very metabolic. Only one cyst seen, 9mm, regular,wrinkled surface. 1-3 CVs, changes location within a singleindividual with movement of cell. 18S-type isolated threetimes: once from sphagnum bog, once from soil, both Borok,Russia (APM); once from pond in San Francisco, USA (DB).Type sequence: 18S: AY884321; ITS2: FJ797445.

Etym. laevus L. on or from the left, where its pseudopodsare commonest.

Cercomonas kiaerdammane Mylnikov, 2002. Figure 4D.Type strain: NY12 (A.P. Mylnikov; Spitzbergen, Norway; 1998).Additional information: Length: 8-13 mm. AF 1.5-2X BL; PF1.25-2X BL. AF movement slow and exploratory. Cell move-ment slow, frequent changes in direction. Finger-like or filosecytoplasmic tail as part of general metabolic tendency of cell.Lamellar pseudopodia almost always present on posteriorand left side of cell. Filose pseudopodia rarer. Extremelymetabolic. 2 CVs in close proximity in posterior half and 1 CVin middle of cell. Multinuclear plasmodia form. Cysts 3.6-7.3 mm, regular to slightly irregular, smooth, medium dense.Type sequence: 18S: FJ790695; ITS2: FJ797449. Furtherobservations: Multiflagellate and multinucleate floating buds,to c. 60 mm diameter, can be formed in older cultures,as bacterial density falls. Buds frequently separate back intotrophic cells. 18S-type isolated twice: once in 2006 fromsummer grassland soil, near Oxford (AH); once in 1998 fromlake water near Ny-Alesund, Spitzbergen, Norway (APM).

Strain AND27 of Lara et al. (2007) has the same 18S-typebut its ITS rDNA sequnce is unknown.

Cercomonas ambigua Howe and Cavalier-Smith sp. nov.Figure 4F. Type strain: WA82 (AH; Oxford; 2006); NIES-2435.Diagnosis: Length: 11-16mm. AF 1.5-2X BL; PF 1.5X BL.Sinuous movement of AF, or flickering at tip. Cell movementslow. Frequent finger-like or filose cytoplasmic tail. Branchedlamellar and finger-like pseudopodia on right, left and poster-ior sides. Very metabolic. 1 central CV. Cysts 5.5-8.2 mm,regular to irregular, sometimes knobbled, very sparse. Isolatedonce from grassland soil. Type sequence: 18S: FJ790696;ITS2: FJ797450.

Etym. ambiguus L. changeable, varying.Cercomonas parambigua Howe and Cavalier-

Smith sp. nov. Figure 4G. Type strain: WA22 (AH; Oxford;2006); NIES-2445. Diagnosis: Length: 11-17 mm. AF 1.3-2XBL; PF 1.3-2X BL. AF movement often slow and probing,occasionally movement restricted to tip. Cell movement quiteslow, pauses frequent. Finger-like and filose cytoplasmic tailsfrequent. Bulbous pseudopodia at anterior and posterior;lamellar pseudopodia most often on left, less often on right,rapidly formed and retracted; filose pseudopodia especially atanterior. Occasional finger-like pseudopodia. Moderatelymetabolic. CVs difficult to observe; usually one in posteriorhalf. Cysts 6.4-7.3 mm, slightly irregular, slightly knobbled,medium. 18S-type isolated three times: twice in 2006 fromsummer grassland soil, and once from winter grassland soil nearOxford (AH). Type sequence: 18S: FJ790697; ITS2: FJ797451.

Etym. para Gk beside, similar to; ambiguus L. changeable,varying.

Cercomonas paraglobosa Bass and Cavalier-Smith sp.nov. Figure 4H. Type strain: 19-3E (DB; Sri Racha, Thailand;2001). Diagnosis: Length: 5-15mm. AF 2-2.5X BL; PF c. 2XBL, tapered. AF usually extended with quite rapid flickersalong the entire length of flagellum, or slower probing. Cellprogression slow. Cytoplasmic tail often as a part of generalcell metaboly; can be filose. Lamellar and finger-like pseudo-podia almost always present, usually at posterior or left side.Very metabolic. Usually 3 CVs in posterior half of cell. Cysts6.0-15.5 (most 6.0-9.1) mm, irregular, sometimes knobbled,very dense. 18S-type isolated three times: once from grass-land soil (KV); in 2006 from grassland soil from near Oxford, UK(AH); from Thai (Sri Racha) rainforest soil (coll. 2001 TCS) (DB).Type sequence: 18S: FJ790698.

Etym. para Gk. Beside, similar to; very similar to Cerco-monas globosa Dujardin, 1841 in size and its active AF, butDujardin’s drawing and descriptions are not detailed enoughto be sure.

Strain AND6 of Lara et al. (2007) has the same 18S-type butits ITS rDNA sequence is unknown.

Cercomonas karpovi Bass, Mylnikov and Cavalier-Smithsp. nov. Figure 5A. Phase contrast and EM micrographsin Karpov et al. 2006. Type strain: C-84 (APM; Borok,Russia; 2001). Diagnosis: Length: 7-12 mm. AF 1.5-2X BL;PF 2-2.25X BL, acronematic. AF movement a constant rapidflickering, ‘casting-out’ movement. Cell usually travelling,though progress often slow and indirect. Slight, infrequentcytoplasmic tail. Bulbous or finger-like pseudopodia quitecommon; lamellar pseudopodia rarer. Not very metabolic. 1CV in posterior tip of cell. Cysts not seen. Type sequence:18S: AY884331; ITS2: FJ797453. Further observations: canswim with slow but direct progress, with periodic fast shakingof posterior end of cell; AF quite steady. Forms plasmodia to�30 mm. Isolated once from a small river.

Etym. after Sergei Karpov, who described it ultrastructurallyin great detail.

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Cercomonas kolskia Bass, Mylnikov and Cavalier-Smithsp. nov. Figure 5B. Phase contrast and EM micrographs inKarpov et al. 2006. Type strain: C-59 (APM; Kol’skii peninsula,Russia; 1989). Diagnosis: Length: 8-13 mm. AF 1.3-2X BL,tapered; PF 1.5-2.25X BL. AF movement: slow waves alonglength. Cell movement very slow, travel very little, often remainin one location. Long, filose or shorter finger-like cytoplasmictails usual. Lamellar, finger-like and filose pseudopodia all overcell. Very metabolic. 1 CV at posterior end. Plasmodia canform. Cysts not seen. Type sequence: 18S: AY884330. Furtherobservations: readily forms meroplasmodia. Isolated once.

Etym. derived from Kol’skii, where sampled.Cercomonas elliptica Bass, Mylnikov and Cavalier-Smith

sp. nov. Figure 5C. Type strain: N8 (APM; Ny-Alesund,Spitzbergen, Norway; 1998); CCAP1910/4. Diagnosis: Length:6-12 mm. AF 1.25-2X BL; PF 1.25-2X BL. AF movementsometimes quite rapid; waves travelling from base to tip. Cellmovement relatively slow; often remain in one location andpivot. Cytoplasm somewhat granular. Very thin filose cyto-plasmic tail, occasionally ‘beaded’, difficult to observe, oftenassociated with PF. Bulbous pseudopodia frequent; lamellarpseudopodia rarer, mostly on left. Not very metabolic. 2-3 CVsin posterior, or left side. Cysts 4.5-6.4 mm, regular, quitesmooth. Isolated once from lake water. Type sequence: 18S:FJ790699; ITS2: FJ797454.

Etym. ellipticus L. elliptical.

Figure 5. Cercomonas clade A1a2. The scale is consgiven for each. A: C. karpovi B: C. kolskia C: C. elliptic

Cercomonas fastiga Bass and Cavalier-Smith sp. nov.Figure 5D. Type strain: 18-3D (DB; garden soil, Oxfordshire;2003); NIES-2438. Diagnosis: Length: 5-11 mm. AF 2-2.5X BL;PF 2X BL. AF movement smooth flickering, often directed upinto medium above the cell. Cell movement: changes indirection frequent, limited distance covered; cell occasionallyrises up into medium and jiggles. Infrequent filose cytoplasmictail. Pseudopodia not observed (in four cells). Not verymetabolic, soft and plastic. Posterior end of cell often tapered.1 CV in posterior half of cell, usually on left. Cysts 4-6 mm,smooth. Type sequence: 18S: FJ790700; ITS2: FJ797455.Further observations: forms plasmodia. Isolated once fromsoil.

Etym. fastigo L. I taper.Cercomonas rotunda Howe and Cavalier-Smith sp. nov.

Figure 5E. Type strain: IVY9G (AH; Oxford; 2006); NIES-2447.Diagnosis: Length: 9-12 mm. AF 2X BL; PF 1-1.5X BL. AFmovement slow and tentative. Cell movement slow, travellinglittle, often pivoting in one location. Filose cytoplasmic tail veryinfrequent. Cell usually round, relatively rigid; slight, infrequentlamellar pseudopodia on left side or posterior. Not verymetabolic. 1 or sometimes 2 found in posterior half, difficult toobserve. Cysts 6.4-8.2 mm, slightly irregular to irregular,smooth, sparse. Isolated once from ivy leaf. Type sequence:18S: FJ790701; ITS2: FJ797456.

Etym. rotundus L. round.

tant for all figures; names and cell length range area D: C. fastiga E: C. rotunda.

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Cercomonas (=Cercobodo) dactyloptera (Skuja 1939)Mylnikov and Karpov 2004. Figure 6A Phase contrastmicrographs and EM in Karpov et al. 2006. Neotype strain:C-43 (APM; Borok, Russia; 1979); NIES-2436. Description:Length: 13-25mm. AF 1-2X BL; PF 1.3-1.5X BL. AF movementrelatively slow sweeping. Progression very slow, often remainsin one location. Finger-like and filose cytoplasmic tail as part

Figure 6. Cercomonas clade A1b2. The scale is consgiven for each. A: C. dactyloptera B: C. braziliensis C:

of general metabolic tendency. Bulbous and lamellar pseu-dopodia all over. Very metabolic. Several CVs throughout cell,except anterior to N. Cysts 8-15 mm, with blunt angularprotuberances giving polyhedral outline, dense, often abun-dant, sometimes forming dense clusters. Type sequence: 18S:AY884334; ITS2: FJ797464. Further observations: cysts varyin size but no distinct size classes. Mero- and holoplasmodia

tant for all figures; names and cell length range areC. lata D: C. effusa E: C. clavideferens.

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both form by cell fusion. Cell cytoplasm granular. Isolatedonce from sewerage water.

Cercomonas braziliensis Howe and Cavalier-Smith sp. nov. Figure 6B. Type strain: B13 (AH; Brazil; 2006).Diagnosis: Length: 13-23 mm. AF 2-3X BL; PF 2-3X BL. AFmovement rapid, flickers left and right and up into medium;movement more probing and exploratory when cell progres-sing. Cell movement rapid and direct when spindle-shaped,slower and probing otherwise. Frequent finger-like and filosecytoplasmic tail. Lamellar pseudopodia all over; finger-likeand filose pseudopodia infrequent; small bulbous pseudopo-dia at anterior and posterior ends. Pseudopodia rapidlyformed, altered and retracted. Metabolic, though less sowhen spindle-shaped. Several large CVs in posterior half ofcell. Cysts 5.5-9.1 mm, quite irregular, smooth, dense toabundant. Cysts formed; circular, contents granular. Formedin great densities. Isolated once from lateritic soil from Brazil(coll. TCS). Type sequence: 18S: FJ790702; ITS2: FJ797463.

Etym. derived from Brazil, the country where found.Cercomonas ‘alexeieffi’ (ATCC50395) and Cercomonas

‘plasmodialis’ AZ-6 (ATCC50418) were not characterizedmorphologically, but according to our phylogeny (Fig. 14)ATCC50395 is expected to be very similar to C. parincurva,and ATCC50418 very similar to C. braziliensis, both describedabove. Lemmermann (1914) described Cercomonas (=Cerco-bodo) alexeieffi as 6-10 mm long, and 5-7 mm wide, with bothanterior and posterior flagella 2.5 - 3X BL. This is so differentfrom C. parincurva that ATCC50395 is probably misidentified,and not alexieffi. Such a morphological discrepancy betweentwo so closely related species would be highly unusual in thecontext of our other data; closely related strains are usuallyextremely similar in appearance, sometimes indistinguishable.

Cercomonas lata Bass, Mylnikov and Cavalier-Smith sp.nov. Figure 6C. Type strain: C-85 (APM; Borok, Russia; 2001);NIES-2442. Diagnosis: Length: 10-30mm. AF 1.5-2X BL; PF1.5-2X BL. AF movement quite slow and probing.Cell movement slow and indirect. Occasional filose or finger-like cytoplasmic tail. Lamellar and finger-like pseudopodia allover cell, most often on posterior and left side; filose andbulbous pseudopodia all over. Very metabolic. 1-2 CVs foundin posterior half of cell. Cysts 10-18 mm, regular to slightlyirregular or angular, blunt angular protuberances producingpolyhedral outline. Type sequence: 18S: AY884325; ITS2:FJ797465. Further observations: can form clusters of 100+rounded cells. Spherical holoplasmodia formed to a diameterof at least 50 mm. Meroplasmodia possibly also formed.Isolated once from a small pond.

Etym. latus L. wide, broad.Cercomonas effusa Vickerman sp. nov. Figure 6D. Type

strain: Beaver-Creek (KV; Arizona, USA; 2001); NIES-2437.Diagnosis: Length 13-25 mm. AF 1.5-2X BL; PF 1.2-2X BL. AFbeats with slow lashing motion. Flagellate changes shapefrequently; shows prominent streaming of cytoplasmic gran-ules in amoeboid movement of body, especially whenprogressing over or through a rough substratum. Foodingested by trawling lamellar pseudopodial extensions ofposterior end of body. Multiple small CVs scattered through-out cytoplasm. Cysts 8-14mm, spherical, smooth to slightlyrough cyst wall. Isolated once from sediment of dried-upfreshwater creek near Montezuma’s Castle, Arizona. Typesequence: 18S: FJ790703.

Etym: effundor L. to stream.Cercomonas clavideferens Vickerman sp. nov. Figure 6E.

Type strain: ATCC50319. Diagnosis: Length 12-24 mm (withtemporary drawn-out tail, up to 40 mm). AF 1.5-2X BL. PF 1.5-1.75X BL. Flagellate capable of gliding or creeping. AF beats

laterally with sweeping stroke during locomotion, PF flexes inturning movements. Spike-like pseudopodia form at anteriorend of body and migrate posteriorly to be absorbed into bodyat tail end along with attached bacteria or small flagellateprey. In creeping (i.e. with body flattened against substratum),body becomes more amoeboid and cytoplasmic stretchingduring amoeboid movements results in a variety of shapesas broader pseudopodia form laterally and intermittently.Several CVs throughout cytoplasm. Cysts 7.5-15.5 mm withcharacteristic wrinkled wall or polyhedral outline; may formclusters in culture. This morphotype was the commonest ingrassland soil samples from Sourhope Research Station,Kelso, UK; isolated in culture on two other occasions (KV).Other isolates of this 18S-type from septic tank, Cambridge,by A. J. Cowling (ATCC 50319), and from Priest Pot, UK by DB(17-12D): these three isolates all have the same ITS1 and 2rDNA sequences. Type sequence: 18S: FJ790704; ITS2:FJ797461.

Etym: clavi L. spikes, deferens L. bearing. Furtherobservations: Meroplasmodia form in 17-12D, but sporadi-cally and unpredictably, sometimes only seenafter intensive observation. Floating buds (holoplasmodia)also form, often as bacterial concentration declines. Thesecould fuse to form an even larger holoplasmodium.

ATCC50319 was represented in Cavalier-Smith and Chao(2003) and Bass and Cavalier-Smith (2004) as a relatively long-branch in clade A. That sequence was suspect because it wasdivergent along the whole length of its 18S, suggesting apseudogene. We re-extracted and re-sequenced this strainand found that the true sequence is very different, much moresimilar to other clade A1b strains than earlier paperssuggested.

Cercomonas sp. Strain C-83: Morphology indistinguishablefrom 17-12D, but distinct in both 18S and ITS rDNAsequences. Only poor quality photos exist, so not named;await re-isolation for description. Isolated once in 2001 from aSphagnum bog, Borok, Russia (APM).

Cercomonas magna Howe and Cavalier-Smith sp. nov.Figure 7A. Type strain: IVY8C (AH; Oxford; 2006); NIES-2443.Diagnosis: Length: 18-38 mm. AF 1.3X BL; PF 1.3X BL. AFmovement fluid, shallow waves passing down length frombase to tip. Cell movement fast and direct. Occasional long,filose cytoplasmic tail. Finger-like and bulbous pseudopodiaall over. Not very metabolic. CV not visible. Cysts 9.1-17.5 mm,irregular, sometimes with blunt angular protuberances, intern-ally asymmetrical, medium-dense. Isolated once from an ivyleaf. Type sequence: 18S: FJ790706.

Etym. magnus L. large, great.Cercomonas pigra Vickerman sp. nov. Figure 7B. Type

strain: CASphII (KV; Mugdock, Glasgow, UK; 2002);CCAP1910/5. Diagnosis: Readily adopts spherical condition(dia. 9-26 mm) in response to temperature change. Length 13-60 mm; AF and PF �1X BL. Movement sluggish, slow, direct,body shape a stocky oval, often with tapering rear end. Lessvariety of shape compared with next species. Pseudopodiabroad and shallow or knob-like. Tiny CVs scattered through-out cytoplasm. Cysts 16-20 mm, spherical with uneven thinouter wall and thick inner wall. Isolated once from sphagnummoss. Type sequence: 18S: FJ790707.

Etym: piger L. lazy, sluggish.Cercomonas sphagnicola Vickerman sp. nov. Figure 7C.

Type strain: CASphI (KV; Dubh Lochan, Loch Lomond, UK;1998); NIES-2448. Diagnosis: Like C. pigra, readily adoptsspherical condition (dia 16-24 mm), but more metabolic ascrawling trophozoite. Length 13-35 mm. AF 1.5X BL, PF 1.5 inmore oval form, less in stretched forms. AF beats slowly,

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progress of gliding flagellate sedate rather than sluggish;in creeping on substratum, lamellar pseudopodia arise onone side, finger-like extensions of lamellae sometimesformed. Multiple small CVs. Cysts 17-23 mm, spherical withsmooth outer wall and deeply-crinkled inner wall; ability

Figure 7. Cercomonas clades A1b1 and A1b3. The scrange are given for each. A: C. magna B: C. pigra C: Cceler.

to form cysts now lost in culture. Isolated once fromdried sphagnum moss. Type sequence: 18S: FJ790708.

Etym: sphagnum+colere L. inhabit.Cercomonas gigantica Mylnikov, 2002. Type strain: NY1.

Type sequence: 18S: AY884320; ITS2: FJ797459.

ale is constant for all figures; names and cell length. sphagnicola D: C. parincurva E: C. vacuolata F: C.

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Additional information: Phase contrast micrograph, draw-ings: Karpov et al. 2006 Figure 3. Isolated once in 1998 fromlake water near Ny-Alesund, Spitzbergen, Norway. Difficult tomaintain in culture.

Figure 8. Cercomonas radiata, Eocercomonas, and Cfigures except C. radiata; names and cell length rangeminuta D: E. minuscula E: E. tribula F: E. echina G: E. rmira.

Cercomonas parincurva Howe and Cavalier-Smith sp. nov. Figure 7D. Type strain: IVY7A (AH; Oxford;2006); NIES-2446. Diagnosis: Length: 18-24 mm. AF 0.75 —1.2X BL; PF 1 - 1.2X BL. AF movement slow. Cell movement

avernomonas n. gen. The scale is constant for allare given for each. A: C. radiata B: E. uvella C: E.

amosa H: Cavernomonas stercoris I: Cavernomonas

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slow, occasionally direct, occasionally with many changes ofdirection. Occasional cytoplasmic tail. Finger-like, bulbous,lamellar and filose pseudopodia predominantly on left sideand posterior end, less often on right side of cell. Pseudopo-dia usually present, often many types at once. Very metabolic;N anterior. CV not visible. Cysts variable, 9-16 mm, regular toslightly irregular, granular, smooth to wrinkled surface. Isolatedonce from ivy leaf. Type sequence: 18S: FJ790709; ITS2:FJ797458.

Etym. incurvus L. crooked or bent; para Gk beside, similarto; as it is similar to Cercomonas (=Cercobodo) incurva(Skuja 1939) Mylnikov and Karpov 2004 in size, granularcytoplasm, and apparent lack of CV, but differs in flagella:-body length ratios and position of N. (Skuja - mid-cell; Ivy7a -anterior).

Cercomonas celer Bass, Mylnikov and Cavalier-Smith sp.nov. Figure 7F. Type strain: C-51 (APM; Rybinsk reservoir,Borok, Russia; 1982); CCAP1910/3. Diagnosis: Length: 13-18 mm. AF 1.5-2X BL; PF 1.5-2X BL. AF movement a flickeringalong length of flagellum, usually on the same plane as the cell.Cell movement quite rapid and direct. Cytoplasmic tail oftensmall and relatively inconspicuous. Infrequent filose and finger-like pseudopodia. Not very metabolic. Several CVs, central or inposterior half of cell. Cysts 7.3-8.2mm, regular, knobbled, verysparse. Type sequence: 18S: FJ790710; ITS2: FJ797460.Further observations: disappears from culture if maintainedabove 15 C. Isolated once from a reservoir.

Etym. celer L. swift.

Cercomonas vacuolata Howe and Cavalier-Smith sp. nov. Figure 7E. Type strain: Wyth3.8 (AH; Oxford;2006). Diagnosis: Length: 16-27 mm. AF 1X BL; PF 1-2X BL.AF movement very rapid and flickering when cell is travelling,though slower and probing when stationary. Cell movementusually very slow; cell often changes shape and directionwhile remaining in one location. Filose and finger-likecytoplasmic tail. Bulbous pseudopodia at anterior and poster-ior ends of cell; occasional finger-like and lamellar pseudo-podia; left side of cell most metabolic. Moderately metabolic.Several CVs in posterior half of cell. Cysts formed; no otherdata. 18S-type isolated twice: once from grassland soil nearOxford (AH); once from dry soil from USA (DB). Typesequence: 18S: FJ790711; ITS2: FJ797457.

Etym. from having numerous vacuoles. Differs by its muchlarger size from C. (=Neocercomonas) jutlandica (Ekelund etal. 2004) Cavalier-Smith and Bass in Karpov et al., 2006,whose 18S sequence is identical or very similar. These twocultures are discussed in detail in the phylogeny section ofResults.

Cercomonas (=Dimorpha) radiata (Klebs 1892) Bass andCavalier-Smith comb. nov. Figure 8A. Strain: xt134 (S. von derHeyden; Priest Pot, UK; 2003). Description: Unstable inculture. Prolonged observation not possible, but our observa-tions are consistent with Klebs’s (1892) original descriptionof C. (=Dimorpha Klebs) radiata, described as 10-14 mm long,5-9 mm wide. Very thin axopodia radiating evenly fromrounded cell in sessile stage, 1-2X cell diameter, often difficultto see under phase contrast. Flagella not always visiblein sessile stage. Klebs’s illustrations are also consistentwith lifestages observed in this culture. Cysts not observed.Type sequence: 18S: FJ790712; ITS2: FJ797466. Furtherobservations: flagellate and immotile stages tend notto overlap significantly in time of occurrence. Isolatedonce as a contaminant in an unrelated culture isolatedfrom Priest Pot. C. rhacodes (Skuja) Mylnikov and Karpov,2004 is similar to radiata, but larger: 27-47 mm long, 10-15mmwide.

Genus Eocercomonas Karpov et al. (2006)

Generally small and compact cercomonads with short flagella,varying in nature of their pseudopodia and propensity to formthem; mostly less metabolic than Paracercomonas. Typespecies: Eocercomonas ramosa Karpov et al., 2006. Figure8G. Type strain: C-80; CCAP1918/1. Type sequence: 18S:AY884327; ITS2: FJ797467. Phase contrast and electronmicrographs in Karpov et al. (2006). Additional information:Length: 5-15 mm. AF 2.5-3X BL; PF 1.5X BL. AF movementquite slow, smooth and languid; movement restricted to distalhalf when travelling. Cell movement: when not in slowlyprogressing motile phase, remains in one location andextends thin, branching, pseudopodia/filopodia, which cananastomose intra- and more rarely inter-cellularly. Cytoplas-mic tail can be 2-3X BL in motile stage; otherwise no morefrequent than production of branching pseudopodia. Very longfilose branching pseudopodia all over cell; finger-like andlamellar pseudopodia less frequent. Very metabolic. 1-3 CVsin various locations. Form a network and pre-plasmodialclusters. Cysts �5(-10?) mm, smooth, infrequent. Furtherobservations: in addition to meroplasmodia, multiflagellateand multinucleate floating buds (holoplasmodia?) can beformed after disturbance of cultures. 18S-type isolated threetimes: in 2001 from a lake in Antarctica (APM); from soil fromBorok, Russia; in 2006 from summer grassland soil nearOxford (AH).

Eocercomonas uvella Bass and Cavalier-Smith sp. nov.Figure 8B. Type strain: 11-7E (DB; El Vedat de Torrente, Spain;2002). Diagnosis: Length: 5-7 mm. AF 1.5-1.75X BL, acrone-matic?; PF 1.5X BL. AF movement quite jerky, fairly rapid,constant waving motion. Cell movement very slow. Nocytoplasmic tail or pseudopodia observed. Not metabolic. 1CV on left side of cell. Cysts 3-6 mm, regular, smooth,infrequent. Isolated once from soil. Type sequence: 18S:FJ790713; ITS2: FJ797468.

Etym. uva L. grape, from shape of cell.Eocercomonas minuta Bass, Mylnikov and Cavalier-Smith

sp. nov. Figure 8C. Phase contrast micrographs in Karpovet al. (2006). Type strain: C-76 (APM; Antarctica; 2001).Diagnosis: Length: 5-6mm. AF 1-1.5X BL; PF 1.5-2X BL. AFmovement smooth, stroking motion. Cell movement indirect,progress limited; often remain in one location probingsurroundings with AF. Cytoplasmic tail. Finger-like pseudopo-dia all over, most often at anterior. Bulbous pseudopodiaperiodically extended and retracted. Filose pseudopodiaoccasionally extend along AF, and form spatulate areas. Softand plastic cell, pseudopodia relatively rare. 2-3 CVs usuallyon the right or left side in posterior half. Cysts unknown. Typesequence: 18S: AY884324; ITS2: FJ797469. Further obser-vations: often grows weakly in culture. Isolated once from afreshwater lake.

Etym. minutus L. small.Eocercomonas minuscula Bass, Mylnikov and Cavalier-

Smith sp. nov. Figure 8D. Type strain: C-73 (APM; near Ny-Alesund, Spitzbergen, Norway; 2001). Diagnosis: Length: 4-10 mm. AF 2X BL; PF 2X BL. AF movement constant wavingalong entire length. Cell movement undulatory and probing;progress slow, changes in direction frequent. Filose cytoplas-mic tail quite often. Finger-like pseudopodia rare; only on leftor posterior. Cell soft, pliable, metabolic, pseudopodia rare.CV usually in the centre on left side. Cysts unknown. Furtherobservations: cells can become rounded, sometimes inclusters, with minimal or no movement. At other times cellsfrequently float a small distance above substrate, movinggently. Often grows weakly in culture. 18S-type isolated twice:

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from garden soil, Oxford (DB); from a Spitzbergen lake (APM).Type sequence: 18S: FJ790714; ITS2: FJ797471.

Etym. minusculus L. smallish.Eocercomonas tribula Howe and Cavalier-

Smith sp. nov. Figure 8E. Type strain: IVY16a (AH; Oxford;2006). Diagnosis: Length: 5-8mm. AF 1-1.5X BL; PF 1.5-2XBL. AF movement relatively slow, probing and whip-like. Cellprogress slow; often stationary and probing. Frequentcytoplasmic tail, often associated with PF. Numerous filosepseudopodia, and branched and finger-like pseudopodia allover cell. Very metabolic. 1 antero-laterally positioned CV.Cysts not seen in some cultures; in others very sparse, regularto slightly irregular, smooth. 18S-type isolated three times;from an ivy leaf, Oxford; and twice in 2006 from summergrassland soil near Oxford (AH). Type sequence: 18S:FJ790715; ITS2: FJ797470.

Etym. tribulus L. for its similarity in appearance to the spinytribulus fruit.

Eocercomonas echina Howe and Cavalier-Smith sp. nov.Figure 8F. Type strain: IVY19 (AH; Oxford; 2006); NIES-2449.Diagnosis: Length: 6-10 mm. AF 1.5X BL and acronematic; PF1.5X BL. AF movement slow. Cell movement slow. Frequentfilose and filose-branching cytoplasmic tail. Numerous finger-like pseudopodia all around cell; filose pseudopodia usual,though constantly extended and retracted; occasional finger-like and slight filose-branching (C) pseudopodia. Metabolic. 1-2 CVs antero-centrally located. Cysts 3.6-5.0 mm, slightlyirregular, slightly knobbled, medium-dense. Isolated once

Figure 9. Cavernomonas mira. Scanning electronmicrograph showing trophic flagellate (ventral sur-face), and two cysts. The two flagella arise from asubapical pit that is continuous with the ventral cleftthrough which food (such as the surroundingbacteria) enters the flagellate’s antrum for ingestion.The emerging posteror flagellum passes through anotch in the right hand wall of the ventral cleft to trailalong the substratum during gliding. The lower cystshows the scar of the cleft after loss of the flagella.Scale bar=5 mm.

from an ivy leaf. Type sequence: 18S: FJ790716; ITS2:FJ797472.

Etym. echinos Gk spiny.

Genus Cavernomonas Vickerman gen. nov.

Diagnosis: Gliding biflagellates with rigid ovoid bodiesdorsiventrally compressed; dorsal surface convex, ventralsurface slightly dished. Flagella emerge from shallow, sub-apical, collared pit on its right hand side, anterior to nucleus.Anterior flagellum beats propelling organism forward; poster-ior flagellum deflected towards rear of organism and trailsbehind, unattached to body. Food ingested into a capaciousventral antrum/pouch via a cleft continuous with the flagellarpit. Pseudopodia not observed. Single CV empties intoantrum. Type species Cavernomonas mira n. sp.

Etym. caverna L. cave, to draw attention to distinctivefeeding mechanism.

Cavernomonas mira Vickerman sp. nov. Figure 8I. Typestrain: Cav-A (KV; Dubh Lochan, Loch Lomond, UK; 1998);CCAP1912/1. Diagnosis: Body oval to circular, 3-8 mm inlength, 3-5mm in width. AF 6.5-10.5mm, PF 12-18mm.Flagellate glides along substratum with wobbling motion,frequently detaching to execute laboured tumbling move-ments through lashing of both flagella until contactingsubstratum with posterior flagellum enables resumed gliding.In turning over and over of body, refraction by thick pellicleresults in flashes of light. Antrum recognisable as elongate,clear, central area extending along anterior 2/3 of body. Cystsroughly spherical, 3.5-6 mm. Isolated once from dried sphag-num moss. Type sequence: 18S: FJ790718; ITS2: FJ797462.

Etym. mirus L. amazing.Cavernomonas stercoris Vickerman sp. nov. Figure 8H.

Type strain: Cav-E (KV; Great Salt Lake USA; 1999); NIES-2434. Diagnosis: Very similar to previous species, but sometimesapparently larger, as shown in Figure 8H; possibly dependent onculture conditions. Cell length 4-9mm. Other morphologicaldifferences not strikingly obvious, but note sequence differencesand organism from very different environments. 18S-type isolatedtwice: once in 1999 from Antelope Island, Great Salt Lake, USAand once from dried dung of American bison, USA, both by KV.Type sequence: 18S: FJ790717.

Etym. stercus L. dung.Remarks: As shown by electron microscopy (Fig. 9 and

transmission electron micrographs not shown — KV), Caverno-monas differ profoundly from all other cercomonads in cellularorganisation: (1) Possession of a thick pellicle/theca (�50 nm)giving rigidity to body. (2) Uptake of food into ventral antrumwhich is not lined by pellicle; pseudopodia from antrum wall maybe responsible for food uptake from antrum into food vacuoles.(3) Cytoskeleton takes form of cortical bands of microtubulesextending from flagellar basal bodies down right and left sides ofbody (cf. intracytoplasmic location of microtubules in othercercomonads). (4) Lack of extrusomes — prominent organellesin most, but not all, other cercomonads. In many respectsCavernomonas shows convergence with apusomonads.

Genus Paracercomonas Karpov et al. 2006(Figs 10—11)

Filose-branching pseudopodia are often present in Paracer-comonas strains, in contrast to Cercomonas. Paracercomo-nas strains are often smaller than those of other cercomonad

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genera. Type species: Paracercomonas marina Cavalier-Smithand Bass in Karpov et al., 2006. Type strain: ATCC50344.

Paracercomonas marina Cavalier-Smith and Bass inKarpov et al., 2006. Type strain: ATCC50344. Type sequence:18S: AF411270; ITS2: FJ797474. Additional information:Isolated in 1990 from sediment from 10-15 meters deep,Castle Harbour, Bermuda. Cysts not recorded. This species isslightly larger than P. minima and P. ekelundi, contrary top. 148 of Karpov et al. (2006).

Figure 10. Paracercomonas clade B1a. The scale is cogiven for each. A: P. filosa B: P. vonderheydeni C: P. minP. pleomorpha H: P. crassicauda.

Paracercomonas (=Cercobodo) minima (Mylnikov, 1985)Bass and Cavalier-Smith comb. nov. Figure 10C. Neotypestrain: SW2 (AH; Oxford; 2006); CCAP1957/3. Description:Length: 5-9mm. AF 1-1.5X BL and acronematic; PF 1-1.5X BL.AF movement quite slow waves along entire length offlagellum. Cell often still and metabolic. Branched, finger-likeor filose cytoplasmic tail always present. Finger-like, bulbous,lamellar, filose-branching pseudopodia almost always presentall around cell. Very metabolic. 1 antero-laterally positionedCV. Cysts 3-5.5 mm, regular to slightly irregular, smooth,

nstant for all figures; names and cell length range areima D: P. producta E: P. compacta F: P. oxoniensis G:

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dense; readily formed. 18S-type isolated 21 times: 13 times in2006 from grassland soil near Oxford, UK; in 2007 fromsoil from Sweden; in 2005 from soil from La Campana, Chile(coll. TCS), all by AH; once from freshwater sedimentand once from soil from New Zealand, from soil fromArgentina (coll. TCS), and from soil from Panama, all byDB; once from grassland soil from Sourhope, UK, and oncefrom soil by KV. Type sequence: 18S: FJ790720; ITS2:FJ797476.

P. minima’s 18S-type is the most frequently cultured of allcercomonads. P. ekelundi (SCCAP C1; 18S sequenceGenBank AY496047) (Fig. 14) Cavalier-Smith and Bass;misidentified as C. longicauda by Ekelund et al. (2004), hasthe same 18S rDNA V4 region sequence as P. minima, butdiffers from P. minima elsewhere in its 18S sequence, so isretained here as a separate species until a culture and ITSrDNA data are available. (SCCAP C1 was given as 4-10 mm inEkelund et al. (2004); this was mis-reported by Karpov et al.(2006), p. 148.) Our other 12 isolates of this 18S-type havealso been ITS2-sequenced, resulting in three ITS2-typesdiffering from each other and P. tenuis (below) in helices 1and 3. All others had identical ITS2 sequences to one of thesethree types. P. minima, P. tenuis, and P. producta (below) formpart of a large cluster of closely related species. Other isolateswith similar morphology but distinct 18S sequences were alsoisolated (e.g. WA10, Fig. 14). Before deciding whether todesignate these as separate species more detailed cultureobservations and experiments are required to determine thenature of differences among them.

Paracercomonas vonderheydeni Bass and Cavalier-Smith sp. nov. Figure 10B. Phase contrast micrographs inKarpov et al. 2006. Type strain: NZ1-5C (DB; New Zealand;2003). Diagnosis: Length: 5-9mm. AF 1-1.5X BL; PF 1.5-2XBL. AF constantly flickering. Cell movement often quite rapidand direct, cell usually travelling. Cytoplasmic tail alwayspresent, often associated with PF. Bulbous, finger-like andlamellar pseudopodia. Very metabolic. 1 CV, located centrallyor laterally mid-cell. Cysts �3.6 mm, regular, smooth, dense;forms clusters which often include many bacteria and floatabove the substrate. Isolated once from New Zealand soil,2003. Type sequence: 18S: AY884342; ITS2: FJ797477.Distinguished from other species by cell movement and cystcharacteristics.

Etym. after S. von der Heyden who collected the soilsample.

Paracercomonas tenuis Bass and Cavalier-Smith sp. nov.Type strain: SA-S (ATCCPRA-61) Cavalier-Smith and Chao(2003) (E. Chao; Fishhoek, Western Cape, South Africa; 2000).Diagnosis: morphologically indistinguishable from P. minima,but has a clearly different ITS2 rDNA sequence in helices 1, 2,and 3. Type sequence: 18S: AF534712; ITS2: FJ797475.

Etym. tenuis L. thin, delicate.Paracercomonas producta Howe and Cavalier-Smith sp.

nov. Figure 10D. Type strain: WA42 (AH; Oxford; 2006); NIES-2454. Diagnosis: Length: 4-9 mm. AF 1-1.5X BL; PF 1-1.5XBL. AF movement usually constant, flickering probing move-ment. Cell movement slow, progress slow, frequent interrup-tions to pause, probe and change direction. Cytoplasmic taillong and very often present. Lamellar, bulbous, finger-like,filose and branched pseudopodia all around cell, almostalways present. Very metabolic. 1 CV, mid-cell, often nearleft or right side. Cysts 3.6-7.3 mm, regular to irregular, smoothto knobbly. Dense to very dense. Can form floating clusterswith bacteria. Isolated once, in 2006 from grassland soilnear Oxford. Type sequence: 18S: FJ790721; ITS2: FJ797478.

Etym. productus L. stretched out, extended.

Strain AND3 of Lara et al. (2007) has the same 18S-type butits ITS rDNA sequence is unknown.

Paracercomonas filosa Bass and Cavalier-Smith sp. nov. Figure 10A. Type strain: 17-1F (DB; PriestPot, UK; 2002). Diagnosis: Length: c. 5mm. AF 1.5X BL; PFnot seen. AF slow waving motion. Cell movement slow‘tentacle-like’ crawling when progressing, though travellinginfrequent; more often remains in one location surrounded byits pseudopodial projections. Occasional cytoplasmic tail.Long, thin pseudopodia emerging from all over cell, oftenspatulate at distal ends. Not very metabolic. Apparently only 1CV. Cysts not seen. Type sequence: 18S: FJ790722; ITS2:FJ797482. Further observations: not vigorous in culture; onlyseen in cultures with minimal enrichment. Isolated once fromPriest Pot, UK (eutrophic pond).

Etym. filum L. thread, string.Paracercomonas compacta Bass and Cavalier-Smith sp.

nov. Figure 10E. Type strain: xt198 (DB; Oxford; 2004).Diagnosis: Length: 4-7mm. AF 1-1.5X BL, tapered; PF 1-1.5X BL. AF movement slow, fairly constant waving. Cellmovement slow and indirect. Cytoplasmic tail usually wideand finger-like. Pseudopodia almost always present; lamellar,filose, and finger-like pseudopodia very often all over cell,usually more than one present at any time. Filose pseudopo-dia occasionally have spatulate ends. Extremely metabolic. 1CV, often near cell posterior. Cysts: no data. Isolated oncefrom Oxfordshire soil. Type sequence: 18S: FJ790723; ITS2:FJ797480.

Etym. compactus L. compact.Paracercomonas oxoniensis Howe and Cavalier-Smith

sp. nov. Figure 10F. Type strain: WA8 (AH; Oxford; 2006);NIES-2451. Diagnosis: Length: 8-16 mm. AF 1-1.5X BL; PF1.5-2X BL. AF movement quite jerky, constant probing; mostmovement in distal half. Cell movement occasionally relativelydirect and rapid, though often remains in one location probingwith AF and extending pseudopodia. Finger-like or very longfilose, or filose-branched cytoplasmic tail. Lamellar, filose,finger-like and filose-branching pseudopodia. Occasionallypseudopodia have spatulate ends. Very metabolic. 1-2 CVsusually in posterior half. Cysts 4-7mm, irregular, not alwayssmooth, often clustered. 18S-type isolated twice in 2006 fromgrassland soil near Oxford (AH). Type sequence: 18S:FJ790724; ITS2: FJ797479.

Etym. ‘of Oxford’ near where soil sampled.Paracercomonas pleomorpha Bass and Cavalier-Smith

sp. nov. Figure 10G. Type strain: 19-5C (DB; Oxford; 2004);NIES-2453. Diagnosis: Length: 5-8 mm. AF 1-1.5X BL,tapered; PF 1-1.5X BL, though often does not extend furtherthan the metabolic cell depending on its shape. AF movementfast flickering; occasionally limited to distal half. Cell move-ment usually fast and direct, occasionally indirect and slow.Posterior pseudopodia extended and withdrawn in ‘walking’motion as cell progresses. Filose and finger-like cytoplasmictail usually present or being extended and retracted. Lamellarand finger-like pseudopodia almost always present, anywhereon cell; occasional filose pseudopodia at anterior of cell.Extremely metabolic. 1 CV found mid-cell or near posteriorend. Cysts not seen, but cells become rounded and inactive,often in clusters. Type sequence: 18S: AY884341; ITS2:FJ797486. Further observations: Cells can aggregate intotight clusters (possibly early-stage holoplasmodia). 18S-typeisolated three times from soil from Oxford (DB).

Etym. pleon Gk multiple morph Gk form, shape.Paracercomonas (=Cercomonas) crassicauda (Dujardin

1836) Bass and Cavalier-Smith comb. nov. Figure 10H.Neotype strain: ATCC50316. Description: Length: 6-10 mm.

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AF c. 1X BL, acronematic; PF c. 1X BL. AF movement regularsweeping unless an object encountered. Cell movementindirect, not rapid. Finger-like or filose cytoplasmic tailregularly extended and retracted. Lamellar, finger-like andfilose pseudopodia. Metabolic. 1 CV in anterior of cell, or lessoften in posterior. Cysts �3.6 mm, mostly regular, smooth, very

Figure 11. Paracercomonas clade B1a continued, andfigures; names and cell length range are given for each.E: P. saepenatans F: P. paralaciniaegerens G: P. anaero

sparse; rounded inactive cells more frequently formed thancysts. Isolated once from soil/litter from polar bear enclosure,Chester Zoo, Chester, England. For ultrastructure see Karpovet al. (2006), who noted that ATCC50316 is possibly notcrassicauda. However, amongst the much larger number ofstrains analysed here it resembles crassicauda sensu Dujardin

Groups B1b and B2. The scale is constant for allA: P. astra B: P. ambulans C: P. elongata D: P. virgariabica.

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more than any other known strain, so we designate it theneotype, concurring with its original identification by H. Smith.Type sequence: 18S: FJ790725; ITS2: FJ797487.

Paracercomonas sp. Strain xt159. 18S sequence: 18S:FJ790726. Isolated once from soil from San Francisco, USAby D. Bass. Culture dead. Only poor quality photos exist, sonot named; await re-isolation for description.

Paracercomonas astra Howe and Cavalier-Smith sp. nov.Figure 11A. Type strain: W89 (AH; Oxford; 2006). Diagnosis:Length: 6-14 mm. AF around 1X BL, tapered or acronematic;PF 1.5X BL. AF movement either beating (not very liquid) orquite still; often hooked to left. Cell movement direct andsometimes quite rapid. Sporadic finger-like or filose cytoplas-mic tail. Lamellar and/or branching pseudopodia at posterior;finger-like most often at anterior. Moderately metabolic. 1 or 2CVs in mid-cell position, to right or left. Cysts 4.5-5.9 mm,slightly irregular, smooth, sparse. 18S-type isolated threetimes: once from grassland soil near Oxford, once in 2007from soil from Sweden, by AH; once from rainforest soil,Panama (DB). Type sequence: 18S: FJ790727; ITS2: FJ797485.

Etym. astron Gk star.Paracercomonas ambulans Howe and Cavalier-Smith sp.

nov. Figure 11B. Type strain: W80 (AH; Oxford; 2006);CCAP1957/4. Diagnosis: Length: 3-7 mm. AF c. 1X BL; PF1-1.5X BL. AF movement a constant flickering and probing.Cell movement a ‘walking’ motion of extending and retractingposterior pseudopodia, or remain in one location, slowlyturning and probing. Cytoplasmic tail as part of generalmetabolic tendency of cell. Lamellar, finger-like and bulbouspseudopodia; very long filose and branched pseudopodiaoften present. Filose pseudopodia occasionally form spatulateends. All types form anywhere on cell. Very metabolic. 1 CVcentral or mid-cell on right, or rarer, mid-cell on left. Cysts 2.7-4.5 mm, regular to slightly irregular, smooth, dense, quiteclustered. 18S-type isolated twice: from Oxfordshire grass-land soil (AH) and from grassland soil from Sourhope, UK (KV).Type sequence: 18S: FJ790728; ITS2: FJ797483.

Etym. ambulare L. to walk, travel.Paracercomonas elongata Howe and Cavalier-Smith sp.

nov. Figure 11C. Type strain: IVY11 (AH; Oxford; 2006); NIES-2450. Diagnosis: Length: 5-12 mm. AF 0.75X BL; PF 1.3-1.5XBL. AF flickers; proximal quarter less active. Cell movementdirect, with a slight pivot on longitudinal axis. Sporadic finger-like, filose and occasionally long, filose-branched cytoplasmictails. Lamellar, bulbous and finger-like pseudopodia all overcell, less frequently at anterior. Not very metabolic. CV central.Cysts 3.6-4.5 mm, regular to slightly irregular, smooth, med-ium-dense. Isolated once from an ivy leaf. Type sequence:18S: FJ790729; ITS2: FJ797484.

Etym. elongare L. to elongate.Paracercomonas virgaria Bass, Mylnikov and Cavalier-

Smith sp. nov. Figure 11D. Phase contrast micrographs inKarpov et al. (2006). Type strain: C-71 (APM); Borok, Russia;2000); CCAP1957/1. Diagnosis: Length: 6-14mm. AF 1X BL,tapered; PF not visible. AF movement slow waving. Cellmovement: usually remain in one location, extending andretracting pseudopodia. Filose cytoplasmic tail; more oftenwhen cell progressing. Long, filose-branching pseudopodia allover cell. Finger-like, lamellar and filose pseudopodia. Occa-sional filose pseudopodia with spatulate ends. Very metabolic.1 CV at anterior end, anterior to N. Only two or three cells canform plasmodial network. Cysts not seen; rounded, barelyactive cells (�5mm diameter) often present in older cultures.Type sequence: 18S: AY884340; ITS2: FJ797473. Furtherobservations: Older cultures show possible meroplasmodiaformed by interconnections of very thin filopodia. Occasional

cell clusters produced (to �55 mm diameter), apparentlywithout cell fusion. In casein-based medium only, holoplas-modia formed within meroplasmodial networks. 18S-typeisolated three times: from soil from Oxford (2006; AH); fromsoil from Borok, Russia (2003; DB), and once in 2000 from asmall river at Borok, Russia (APM).

Etym. virga L. twig, sprout.Paracercomonas paralaciniaegerens Bass and Cavalier-

Smith sp. nov. Figure 11F. Type strain: 31-1C (DB; Oxford;2004); NIES-2452. Diagnosis: Length: 9-18 mm. AF c. 1X BL,tapered; PF c. 1X BL. AF movement fluid constant flickering;finger-like pseudopodia often extend a small way along AF.Cell movement slow and indirect, changes of direction andpivoting frequent. Frequent filose or finger-like cytoplasmictail, usually present. Lamellar, filose, and most commonly,finger-like pseudopodia very common, all over cell, constantlyextending, altering and retracting. Very metabolic. 1-3 CVsfound in various locations. Cysts 5.5-8.2 mm, regular to slightlyirregular, smooth, dense. Isolated once from soil fromOxfordshire, UK. Type sequence: 18S: FJ790730.

Etym. culture similar to Krassilstschik’s 1886 description ofCercobodo laciniaegerens in that the AF arises near a short‘snout’. Other details possibly inconsistent. Para Gk beside,similar to; lacinia L. protuberance; egerans L. carrying or,bearing out.

Paracercomonas (=Cercomonas) metabolica (Zhukovand Mylnikov, 1987) Karpov et al., 2006. Strain: CS-9. (sameisolate as Cercomonas metabolicus HFCC88 Wylezich et al.2007). Phase contrast micrographs in Karpov et al. 2006.Additional information: Length: 5-15 mm. AF 2-2.5X BL,acronematic; PF 1.5-2.5X BL. Flat lobopodial pseudopodia allover cell, except when swimming, when the spindle-shapedcell with condensed cytoplasm revolves on its longitudinalaxis. 1-2 CVs, 1 at anterior of cell, and occasionally a secondin posterior. Cysts not seen. Type sequence: 18S: AY884339.Further observations: very metabolic; forms plasmodia.Isolated once in 1980 from soil, Borok, Russia (APM).

Paracercomonas saepenatans Vickerman sp. nov. Figure11E. Type strain: CA5HKV (KV; Sourhope, Kelso, UK; 1999);CCAP1957/2. Diagnosis: One of very few known cercomo-nads that, in addition to gliding/creeping along substratum,can detach and become free swimming but is still capable ofdirectional locomotion. In gliding phase, body (8-14 mm)broadly elliptical with pointed anterior and posterior. AFemerges from conical projection at anterior tip; 2-3X BL. PFattached along 2/3 body length, 2-3X BL. Flagellate glidessedately along substratum, flicking end of AF. Speed ofbeating increases dramatically as it moves into swimmingphase, adopts a subspherical body form, and eventuallydetaches PF from substratum to swim freely along a helicalpath. Tongue-like pseudopodia, sometimes forked, formedwhen gliding flagellate settles in body contact with substra-tum. Large, single CV empties at base of the anterior cone.Spherical cysts 7-11 mm, smooth-walled. In subculture,swimming forms appear after 3 days. Distorted, elongate,poorly motile, non-swimming forms mark the decline phase.Isolated once from grassland soil. Type sequence: 18S:FJ790731.

Etym. saepe L. often; natans L. swimming.In its ability to swim, this species resembles Cercomonas

(=Cercobodo) heimi (Hollande, 1942), but comparison with thelatter is difficult as Hollande’s description is based on stainedor highly compressed living specimens. C. heimi wasdistinguished by its twin contractile vacuoles rather than asingle large vacuole, emptying anteriorly; a prominent anteriorcone was not mentioned or depicted.

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Paracercomonas sp. Strain Panama67 (DB, 2003). 18S:FJ790732. Notes: Length: 5.5-8.5 mm. AF 1-1.5X BL; PF1-1.5X BL. AF movement quite slow sweeping arc. Cellmovement slow indirect travelling. Posterior pseudopodia give‘walking’ effect. Occasionally pivot in one location. Cytoplas-mic tail. Pseudopodia all over cell. Very metabolic. Cysts: nodata. Isolated from rainforest soil, Panama.

Paracercomonas sp. Strain Panama89 (DB, 2003). 18S:FJ790733. Notes: Length: 5.5-8.5 mm. AF c. 1X BL; PFunknown. Cell movement slow. Pseudopodia. Very metabolic.Cysts: no data. Isolated from rainforest soil, Panama.

Paracercomonas sp. Strain Panama101 (DB, 2003). 18S:FJ790734. Notes: Length: 5.5-7.5mm. AF 2- 3X BL; PFunknown. AF movement fairly constant sweeping and flickering.Cell movement: slow travel, often wriggling in one location.Cytoplasmic tail unknown. Pseudopodia all over cell. Verymetabolic. Cysts: no data. Isolated from rainforest soil, Panama.

Paracercomonas sp. Strain Panama107 (DB, 2003). 18S:FJ790735. Notes: Length: 10-15.5mm; AF c. 1.5X BL; PF 1-1.5XBL. AF movement quite constant, rapid sweeping and flickering.Cell movement quite rapid, often travelling. Occasionally cellremains in one location, projects up into medium and jiggles.Cytoplasmic tail. Bulbous, finger-like and filose pseudopodiarapidly formed, altered and retracted. Very metabolic. Cysts: nodata. Isolated from rainforest soil, Panama.

Paracercomonas sp. Strain Panama61 (DB, 2003). 18S:AY884336. Notes: Length: 8.5-11.5mm. AF 1.5X BL; PF c. 2XBL. AF movement flickering, and quite whip-like. Cell move-ment: often remains in one location, pivoting and waving AF.Cytoplasmic tail unknown. Pseudopodia unknown. Metabolic.Cysts: no data. Isolated from rainforest soil from Panama.

Paracercomonas sp. Strain xt181 (DB, 2003). 18S:FJ790736. Only poor quality photos exist so not named;await re-isolation for description. Isolated from wet rainforestsoil, Panama. A closely related strain (AND18) isolated by Laraet al. (2007) formed cysts.

Paracercomonas sp. Strain Panama83 (18S: FJ790737)(=Cercomonas sp. Tempisque (Cavalier-Smith and Chao2003), AF411271). Notes: Length: 8.5-11mm. AF c. 1X BL;PF c. 2X BL. AF movement rapid constant flickering. Cellmovement can be quite rapid, with moderately frequentchanges of direction. Cytoplasmic tail. Pseudopodia all overcell. Very metabolic. Cysts not seen. Isolated twice: once in2003 from rainforest soil, Panama (DB); and once from theTempisque river, Costa Rica (EC, December 1996).

Paracercomonas anaerobica Cavalier-Smith and Bass sp.nov. Figure 11G. Type strain: ATCC50367 (T.A. Nerad; site anddate unknown). Described in ATCC catalogue as ‘Cercomonassp.’. Diagnosis: Length: 5-8mm. AF stubby; PF 1.5X BL. AFmovement quite rigid, usually held perpendicular to the cell’santerior-posterior axis and sweeps. Cell movement slow andsteady, progress quite direct. Cytoplasmic tail. Fine finger-likepseudopodia most often at posterior end; less frequent bulbousand lamellar pseudopodia at posterior end. Somewhat meta-bolic. 2 CVs, 1 in anterior half, 1 in posterior. Occasionally 3present. Cysts not seen, but cells can become rounded andinactive. Isolated once. Type sequence: 18S: AF411272; ITS2:FJ797481. Further observations: grows in both low andstandard oxygen levels. See electron microscopy below.

Etym. anaerobic because of its characteristic ability to growin anaerobic conditions.

Neotypifications

Three species require neotypification in order to stablise theirnomenclature. The same interpretation of the Zoological Code

of Nomenclature (ICZN) applies to all of them (InternationalCommission on Zoological Nomenclature 1999):

Cercomonas (=Cercobodo) dactyloptera (Skuja, 1939)Mylnikov and Karpov, 2004. Type strain C-43: NIES-2436.Paracercomonas (=Cercobodo) minima (Mylnikov,1985) comb. nov. Type strain SW2: CCAP 1957/3.Paracercomonas (=Cercomonas) crassicauda (Dujardin,1836) comb. nov. Type strain ATCC50316.Conditions of article 75.3 of ICZN for valid neotypification

are met thus:

1.

We designate the strains listed above as the hapanto-type culture and neotype for the associated species forthe express purpose of clarifying the taxonomic statusof that species.

2.

Each species neotypified can be distinguished from allother species by its rDNA sequence (here we useV2+V4+V5+V7 18S plus ITS2 rDNA, where available).Any strains with an unambiguously different rDNAsequence (as defined in the Methods) are to beregarded as separate species. Those with identicalrRNA are to be regarded as the same species (unlessother reliable evidence indicates that they should beplaced in separate sibling species). Descriptions of theneotype strains are given in the previous section butwithout rRNA sequences these descriptions are mostlyinsufficient to recognise them unambiguously.

3.

The neotype of each species is the culture specified, incombination with its rDNA sequence as specified andlight micrographs listed for each strain. Thus recogni-tion of the neotypes designated is assured.

4.

There are no type specimens or type micrographs forthe species for which we are designating neotypes.Thus no holotype ever existed for them.

Electron Microscopy of Paracercomonasanaerobica

The cytoplasm is densely packed with ribosomes,numerous pale ellipsoid granules, presumablysecretory (Fig. 12A, E), and a large dorsal regionof rough ER cisternae, which are much moreextensive than in most protozoa, possibly highlydeveloped for manufacturing secretory proteinsfor the ellipsoid granules. Phagocytosed bacteriawere present in food vacuoles; one micrographshows that that these are caught by extremely thinlamellipodia (Fig. 12E). Mitochondria with tubularcristae and exceptionally dense matrix are numer-ous (this culture was grown without specialanaerobic precautions). The nucleus is highlyelongated and drawn out to a slender point towhich the two centrioles, embedded on a ribo-some-free matrix, are attached. The posteriorcentriole is closer to the nucleus and bears a long

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recurrent flagellum that in places appears to be ina distinct ventral groove (Fig. 12G); it is associatedwith at least two microtubular roots: a simplelateral one of two microtubules, and a complexone (also proximally with two microtubules)associated with dense fibrous material betweenit and the anterior centriole. The two centriolesdiverge at about 45 degrees and are mutuallyattached by about two cross-striated connectors.Though we did not obtain serial sections, thecentriolar roots are obviously markedly simplerthan in clade A cercomonads (Karpov et al. 2006).Also unlike clade A cercomonads, the long axes ofthe two centrioles are not approximately in oneplane, but very skewed; moreover the posteriorcentriole is apparently not docked onto theanterior one but their bases are more symmetri-cally arranged. In the transitional region of theflagellum there is an axosome at the distal endand a hub-lattice structure at its basal end (seeCavalier-Smith et al. 2008); the relative densities ofthe components of the hub-lattice structure differfrom those of clade A cercomonads (see Discus-sion).

Taxonomic and Nomenclatural History ofCercomonads

Following the pioneering work of Dujardin (1841)(outlined in the Introduction) Stein (1878) describedseven species, three previously described byDujardin (1841): Cercomonas longicauda, C. cras-sicauda, and C. lobata, although Stein’s drawingsdisagree with those of Dujardin. Three weremisidentified as cercomonads: C. ramulosa, C.muscae-domestica, and C. termo (Monas termoEhrbg.), and clearly represent three differentgenera. The last, a probable true cercomonad C.obesa, corresponds to none of our isolates.

Krassilstschik (1886) established a new genus,Cercobodo, as a distinct group of amoeboflagel-lates that form pseudopodia as Cercomonasspecies do, but stated that in contrast toCercomonas, Cercobodo species were biflagel-lates. He described a single species — C.laciniaegerens — and designated it as type. Alsorejecting the genus Cercomonas, Klebs (1892)noted that Dujardin’s genus had not been suffi-ciently defined and descriptions were incomplete;because Dujardin’s description was not clear onflagellar number, Klebs used the genus nameDimorpha (Gruber) for four biflagellates: Dimorphaovata Klebs, D. radiata Klebs, D. longicauda(Dujardin) Klebs, D. alterans Klebs. This genusname was briefly supported by a few authors,

including Meyer (1897), who described two morespecies, D. bodo and D. digitalis. The reasonsgiven by Krassilstschik and Klebs for rejectingDujardin’s name are not nomenclaturally sound;moreover the name Dimorpha was preoccupiedand invalid for protozoa (see Bass et al. 2009a).Nonetheless, Krassilstschik’s Cercobodo hadmany advocates as the appropriate name forpresent day cercomonads (Hanel 1979; Hollande1942; Klug 1936; Lemmermann 1910, 1914; Skuja1939, 1948, 1956; Skvortzow 1932) and was thegenus name under which the majority of cerco-monad species was described. However, since itwas recognised that Dujardin divided monads intotwo groups: those with a single flagellum, andthose with ‘‘plusieurs filaments ou appendices’’and Cercomonas was placed in the latter, and thatthe posterior flagellum could easily have beenoverlooked, rejection of Cercomonas on the basisof flagellar number was deemed unsound (Mylni-kov and Karpov 2004), and Cercomonas is nowaccepted as the correct genus name (Karpov et al.2006; Mylnikov and Karpov 2004; Patterson andZolffel 1991). Mylnikov and Karpov (2004) cemen-ted the concept of cercomonads as more or lessamoeboid biflagellates, and list the describedspecies and their authorities. The subsequent fateof the name Cercobodo is discussed furtherbelow.

A significant contribution to cercomonad tax-onomy was made by Ekelund et al. (2004), whowere the first to argue that Cercomonadidaeneeded subdividing into more than one genus.However, their misidentification of the culture theydesignated as neotype for Cercomonas long-icauda Dujardin (1841) (this species being desig-nated as type by Fromentel (1874)) means thattheir assignations of the new generic nameNeocercomonas to clade A cercomonads andCercomonas to clade B were invalid, as explainedby Karpov et al. (2006). Although it is impossible tounambiguously identify the species representedby Dujardin’s (1841) original description, the strainclosest to this description was C. longicaudaCCAP 1910/2, which was designated as neotypeby Karpov et al. (2006), with the consequence thatclade A1 cercomonads, to which this strainbelongs, must be Cercomonas.

Summary of Nomenclatural Innovations

In this paper, we have named 56 species de novo— 35 Cercomonas, five Eocercomonas, twoCavernomonas gen. nov., and 14 Paracercomonas.

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In addition we assigned 10 published names toour new cultures as follows. Cercomonas: C.longicauda Dujardin (1841), C. (=Cercobodo)dactyloptera (Skuja 1939), C. kiaerdammane andC. gigantica Mylnikov (2002), and C. (=Dimorpha)radiata (Klebs 1892). Eocercomonas: E. ramosaKarpov et al. (2006). Paracercomonas: P. (=Cerco-bodo) minima (Mylnikov 1985), P. (=Cercomonas)metabolica (Zhukov and Mylnikov 1987), P. (=Cer-comonas) crassicauda (Dujardin 1836), andP. marina Karpov et al. (2006). In five cases whereour descriptions were close to previously pub-lished ones and did not disagree on any definitivepoint, yet there was insufficient information for usto adopt the earlier name, we prefixed our specificepithet with ‘par(a)’: Cercomonas paraglobosareferring to Cercomonas globosus Dujardin(1841), Cercomonas paravarians and Cercomonasdiparavarians referring to Cercobodo varians Skuja(1948), Cercomonas parincurva referring to Cer-cobodo incurvus Skuja (1939), and Paracercomo-nas paralaciniaegerens referring to Cercobodolaciniaegerens Krassilstschik (1886). We analysedas many strains deposited as putative cercomo-nads in culture collections as possible; the nomen-clatural results are in Supplementary Table S2.

Agitata gen. n. and ‘Cercobodo agilis’(CCAP 1901/1)

We describe below the previously misidentifiedbiflagellate ‘Cercobodo agilis’ (CCAP 1901/1)(Fig. 13) as a new species, Agitata tremulans.The name Cercobodo has a chequered history,arising long after Dujardin (1841) defined Cerco-monas as amoeboid flagellates with an anteriorflagellum and a posterior extension (tail) to the cellvarying in length and thickness. As noted above,Krassilstschik (1886) created Cercobodo, forsimilar flagellates more obviously with two flagella,even though Dujardin’s drawings are evidently oftypical cercomonads that invariably have a poster-

Figure 12. Electron micrographs of random sections ofthrough the anterior end of the cell, showing nucleusextrusome (e), rough endoplasmic reticulum (RER), anseparate cell. C. Tip of the cell in A enlarged to showanterior) attached to the nuclear apex (N) by ribosome-fflagellum, showing the basal striated connector (c) to theendowed with ellipsoidal granules (g); from its irregulawrapping round a bacterium (B). F. Transverse section thshowing the simple microtubular root (r) and the comenvelope. G. Longitudinal section though posterior flag

ior flagellum beneath, or extending beyond, theirprotoplasmic tail (whence came the prefix Cerco— Greek for tail). While it is now accepted thatCercomonas has priority for such typical cerco-monads, some authors have sought to retain bothnames, using Cercomonas for flagellates wherethe posterior flagellum adheres to the body right tothe tail and Cercobodo for Cercomonas-likeorganisms whose posterior flagellum is lessadherent and moves independently of the cellsurface, e.g. Dimastigamoeba (=Cercobodo) agilis(Moroff 1904) and Cercobodo vibrans Sandon,1927, the latter also differing from Cercomonas bya ‘greater distinctness of the amoeboid andswimming stages’ (Sandon 1927). Previously wethought that such independence also occurred insome true Cercomonas species (Karpov et al.2006). However, our far more thorough observa-tions during the present study show that althoughthe posterior flagella of some cercomonad strainsmay temporarily become detached from the bodythey are never consistently in one state or theother. In Cercomonadida as presently constituted,only Cavernomonas has a posterior flagellum thatdoes not adhere to its body at all, and it would nothave been confused with Cercomonas/Cerco-bodo in the past because it is entirely non-amoeboid. Despite its identification as ‘Cerco-bodo agilis’, CCAP 1901/1 clearly differs fromboth Cercobodo laciniaegerens Krassilstschik inshape and size, and Dimastigamoeba (=Cerco-bodo) agilis (Moroff 1904), because of differencesin pseudopodial size and shape and the shape ofthe cell in active gliding mode. Thus it is a newspecies wrongly identified as C. agilis. Although itis not a cercomonad phylogenetically (Bass et al.2005), it somewhat resembles some cercomonadsbecause its posterior gliding flagellum can adheretemporarily to its body. However, this could bethe ancestral state for most or even all ofthe subphylum Filosa and does not imply closeaffinity to cercomonads. Until Cercobodo laciniae-gerens, the type species, is rediscovered and

Paracercomonas anaerobica. A. longitudinal section, mitochondria (m), an engulfed bacterium (B), and an ellipsoidal granule (g). B. RER cisternae in a

the obliquely sectioned centrioles (pc, posterior; ac,ree matrix. D. Longitudinal section through posteriorobliquely sectioned anterior centriole. E. A cell richlyr surface a long thin lamellipodium extends and isrough posterior centriole in its ribosome free matrix,plex root (arrow); ac=anterior centriole; N=nuclearellum in its ventral groove.

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Figure 13. DIC photomicrographs of Agitata tremu-lans sp. n. Cell length 6-11 mm.

510 D. Bass et al.

characterized phylogenetically, the name Cerco-bodo should not be used for any other protozoa; ifC. laciniaegerens proved to be a cercomonad, thename Cercobodo would never become availablefor non-cercomonads like CCAP 1901/1. Thisstrain is now classified as a pansomonad, related

Figure 14. Bayesian 18S rDNA phylogeny of all uniquefour cercomonad genera are represented, rooted on a(Pansomonadida). 101 sequences, 1596 positions. Bayethan 0.75; ML booststraps given where greater than 50%threshold. Black blobs indicate support values of 0.95/

to Aurigamonas (Vickerman et al. 2005). It isgenetically and morphologically sufficiently dis-tinct from Aurigamonas to merit a new family,genus, and species:

Family Agitatidae Cavalier-Smith and Bassfam. n. Diagnosis: Ovoid biflagellate anisokontfreshwater phagotrophs which glide agitatedly ontheir longer posterior flagellum; distinguished fromglissomonads (Howe et al. 2009) by 18S rDNAsequences that robustly group them more closelywith Aurigamonas than with glissomonads (seeFig. 14 and the phylogenies in Cavalier-Smith et al.2008 and Howe et al. 2009). Cells not veryamoeboid, but with short rounded to pointedprojections sometimes emerging from any pointon the cell. Type and sole known genus: AgitataCavalier-Smith and Bass gen. n. Diagnosis: withcharacters of the family Agitatidae. Type species

Agitata tremulans Cavalier-Smith and Basssp. n. Figure 13. Type strain: CCAP 1901/1.Diagnosis: as for the family, plus: Length: 6-11mm. AF 1.75-2.25X BL (acronematic; lengthafter acroneme varies); PF 2.25-2.75X BL. Flagellaemerge at anterior end of cell; both AF and PFactive; PF less so, often trails; AF movement rapidflickering along entire length. Cells travel very little,but usually very active: movement vibratory andundulatory; often projecting up into medium andwriggling energetically waving both AF and PF.Filose, finger-like, and more rarely lamellar cyto-plasmic tail, usually associated with PF; can bevery long. Cell anterior sometimes forms project-ing ‘beak’, frequently not attached to the sub-strate. Filose, finger-like, lamellar and bulbouspseudopodia frequently extended and retracted.Cell very metabolic, often round, elongated orspindle-shaped. Nucleus central; 1 CV, usually inposterior half, often nearer left than right. Differsunambiguously from Aurigamonas by absence ofhaptopodia, flagella:body length ratios, and modeof locomotion (Vickerman et al. 2005).

Etym.: from L. agitare to agitate and tremulanstrembling, because of its characteristic move-ment.

This flagellate was misidentified prior to beingplaced in CCAP, as it is not Cercobodo agilis,a species first described as Dimastigamoebaagilis by Moroff (1904) and later transferred to

18S-types derived from cultured cercomonads. Allfilosan outgroup, including Agitata tremulans sp. n.sian posterior probabilities (PP) shown where greater. Both support values are shown if either exceeds its

95% or above.

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Cercobodo Krassilstschik by Lemmerman andthen to Cercomonas by Mylnikov and Karpov(2004). A new genus was necessary for C. agilis asnone of these three names is applicable. Mostflagellates once named Cercobodo are currentlydispersed among Cercomonas, Eocercomonasand Paracercomonas; Cercobodo is a juniorsynonym for the former broader genus Cercomo-nas that embraced all Cercomonadidae (Karpov etal., 2006). Applicability of the name Cercobodoremains in some doubt as C. laciniaegerens(Krassilstschik 1886) has not been cultured andsequenced; however its pseudopodial morphol-ogy makes it likely to belong in Cercomonadidae,which do not group with C. agilis on trees, andthere is no reason to think that it is more closelyrelated to the sequenced CCAP 1901/1. Phyloge-netic evidence is strong that Agitata does notbelong in Cercomonas or Cercomonadidae (Fig.14; Cavalier-Smith et al. 2008; Howe et al. 2009).D. agilis was not the type species of that genus.As Dimastigamoeba Blochmann (1894) is a syno-nym for Cercomonas (Patterson and Zolffel 1991)it is equally inapplicable to this non-amoeboidflagellate.

Phylogeny

Figures 14 and 15 show that existing cercomonadsequences and all those generated by this study(both culture- and environment-derived), plusrepresentatives of all cercomonad 18S-typespresent in GenBank as of December 2008, fallinto the two major, previously described cerco-monad clades, A and B (Bass and Cavalier-Smith2004; Cavalier-Smith and Chao 2003; Karpovet al. 2006). Within these two clades importantsubdivisions occur. Clade A was divided intoCercomonas and Eocercomonas on the bases ofrobust phylogenetic separation and strong differ-ences in centriole and centriolar root structures(Karpov et al. 2006). Here we show that membersof these two genera differ significantly in live

Figure 15. Bayesian 18S rDNA phylogeny representinsingleton lineages detected from culture- and environmthis paper, and all cercomonad 18S-types present inpositions. Bayesian posterior probabilities (PP) shown wgreater than 50%. Both support values are shown if eitindicate support values of 0.95/95% or above. Symbolssequences from GenBank and our unpublished sequencthe lineage indicated. Black blobs=culture-derived slabels in bold italics indicate clades not represented by

cultures, Cercomonas being generally larger withrelatively longer flagella, and with more metaboliccells than Eocercomonas. Since 2006, we havesequenced two strains grouping as sister toEocercomonas, which are smaller still with rigid,non-amoeboid bodies and flicking anterior fla-gella, recalling large glissomonads (=‘heteromi-tids’ pro parte: see Howe et al. 2009), which aredifferent enough from Eocercomonas morpholo-gically and phylogenetically to merit a new genus,Cavernomonas.

Clade A1 is also easily divisible into two smallerclades, A1a and A1b. Although A1b is not oftenrobustly supported in 18S rDNA trees, its mem-bers are significantly larger than those in A1a,usually with highly metabolic cells with granularcytoplasm in which contractile vacuoles are eitherdifficult to see or absent. Clade A1b includes thelargest described cercomonad — C. gigantica(Mylnikov 2002). There are two exceptions tothese generalizations about clade A1b: Cercomo-nas celer (clade A1b3) and C. radiata (A1c). Theformer is smaller than strains in clades A1b1 andA1b2 (although still appreciably larger than mostof clade A1a), and does not grow in culturesabove 15 1C; the latter is strongly dimorphic,having an immobile, heliozoan-like stage withaxopodia radiating from all around the cell, and aclade A1a-like flagellate stage which was gener-ally rare in our cultures. This species grew poorlyin culture, only occurring reasonably densely whenthe bacteria on which they presumably feed werevery dense, and not persisting for many days. Thephylogenetic position of C. radiata is uncertain,although with the environmental sequences7E.IRDBW and 17-4.9.SA in Figure 15, it forms aclade which we designate A1c, pending moredata.

The other member of clade A1b3, C. vacuolata,is also of interest in that its 18S rDNA sequence isvery close to that of Cercomonas jutlandica ofEkelund et al. (2004), the V4 region being identical,but with differences elsewhere in the molecule —both sequences contain apparent errors, the

g all Cercomonas and Eocercomonas clades andental gene library-derived sequences described in

GenBank in December 2008. 100 sequences; 1441here greater than 0.75; ML bootstraps given where

her exceeds its threshold. Black blobs on branchesto the right of branch labels indicate the numbers ofes that are most closely related, but not identical, to

equence; squares=environmental sequence. Cladecultured strains (Fig. 14).

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sequence of N. jutlandica in GenBank (AY496048)is missing the left-hand end of the molecule, butthe variable 18S rDNA regions are generallyidentical between them. Therefore, it is difficultto know whether these sequences representexactly the same 18S-type. If it is the same, thenITS rDNA sequences of each should be com-pared; if they differ then it is possible that theserepresent closely related species which differmorphologically to an unusual degree:C. jutlandica is reported as being significantlysmaller (10-16 mm) than C. vacuolata (16-26.5mm).

The strongly supported clade A2 comprises twogenera, both also strongly supported by allmethods: Eocercomonas, established by Karpovet al. (2006), and the new genus Cavernomonas.A2’s sister position to Cercomonas is also stronglysupported and recovered in all good trees.Members of clade A2, including Cavernomonas,have not been found in any other publishedenvironmental libraries.

Clade B cercomonads (Paracercomonas) aregenerally smaller and more compact than Cerco-monas (though sometimes still highly metabolic),with less flexible movement of the anteriorflagellum. The morphologies of Clade B1a andGroup B1b are not very different (although theknown B1b species are more likely to havethin/filose projections all around the cell). It isinteresting to note that six B1b strains (B1b-Pan)were isolated in a single four-week visit to Panama(and died during the journey back), only one ofwhich (P. heimi) has been isolated in Oxfordfrom other global samples in over five years.As the same isolation and culturing techniqueswere used in both Oxford and Panama, thisdominance of Panamanian strains was probablycaused by local abundance of these strainsand/or other culturing variables (e.g. temperature)that were unavoidably different between thosetwo places. The 18S-types from Panama arenot endemic to that region, as shown in Basset al. (2007). Clade B1c (not designated in Karpovet al. 2006) is represented by two closely relatedcultures, both probably dead. The light micro-scopy description of one of them, AND18, inLara et al. (2007), is 5-10mm long, with filopodiaformed from any part of the cell; posteriorflagellum 1-1.5X BL, and with a refractile granulewhich was also visible in the cysts; isolated fromsoil.

Group B2 comprises only three cultured strains(Panama83 has the same 18S-type as a now deadstrain isolated from the Tempisque River, CostaRica in 1996). Neither 18S-type has ever been

recovered in any of our cercozoan-specific envir-onmental gene libraries, nor to our knowledge inany published environmental survey. Panama83survives only in a poor quality video and is anovoid-lanceolate, swiftly gliding cell with a flicker-ing anterior flagellum, 0.5—1X BL; this descriptionis concordant with that noted for the Tempisquestrain. P. anaerobica is quite different morpholo-gically, as described above (and has a verydifferent 18S rDNA sequence).

The types of pseudopodia shown in Figure 1show a degree of phylogenetic patterning. Lamel-lar pseudopodia were not seen in any strains ofEocercomonas (except long-branched member E.ramosa, C-80). Bulbous pseudopodia were verycommon in Cercomonas clade A1b strains, andless common in the others. Filose and finger-likepseudopodia were present fairly commonly in allgenera (except Cavernomonas). Filose-branchingpseudopodia were most often seen in Eocerco-monas and Paracercomonas strains, but only oneCercomonas strain.

Figure 15 shows a comprehensive representa-tion of 18S-types from both cultures and environ-mental gene libraries — not all 18S-types recov-ered by this study are shown for reasons ofpresentation and analytical expedience, but all arerepresented by a close or robustly sistersequence. All 18S-types present in GenBank asof December 2008 are represented, however,even if only by symbols to the right of the branchlabels. Three clades — A1a3, A1b4, and B3,labeled in bold italic — are represented only inenvironmental libraries, and have never beenisolated into culture. Those in clade A are similarenough to culture-derived sequences to suggestthat additional culturing effort would quite easilylead to their isolation. However, clade B3 is ofparticular ecological and morphological interestdue to its basal position in the Paracercomonasclade, and its phylogenetic position relative toP. anaerobica. Environmental sequences wereonly generated for clade A in this study, and thesefar outnumber those in GenBank. A similarenvironmental investigation of Paracercomonas,Cavernomonas, and Eocercomonas would likelyalso reveal many novel lineages.

The actual number and coincidence of culture-and environmental library-derived sequences isshown in Supplementary Table S1. Only 19 out of53 cultured Cercomonas and Eocercomonas 18S-types were also found in environmental librarieseven after sampling over 50 libraries (although notall culture-derived 18S-types are easily amplified bythe ‘clade A-specific’ primers — see supplementary

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Table 1). Conversely, 104 environmental 18S-typeshad no cultured representatives (not all datashown). The two most frequently cultured clade Aspecies (Cercomonas paravarians and C. media)were each detected independently six times inenvironmental libraries, but the two most frequentlydetected environmental sequences (found in 15 and10 libraries: Cercomonas fastiga and C. hiberna)were cultured only once and twice respectively.

Discussion

Cercomonad Biodiversity

In this paper we have shown the range ofcercomonad morphological diversity both withinand among strains (defined as 18S-types). From atotal of 165 cultures we resolved 66 species, 56 ofthem novel. Only 10 strains could be confidentlyassigned to previous descriptions; this is partlybecause some of these descriptions provideinsufficient bases for making such decisions, butmostly because cercomonad diversity is veryextensive and we believe that small but consistentdifferences between strains are important indistinguishing species. However, it is undeniablydifficult to describe any particular strain ade-quately, both because of their short-termmorphological plasticity and their longer term‘behavioural’ diversity (e.g. propensity to formcysts, plasmodia, differing resting forms, etc.).Some strains, when re-examined after months ofinoculation, have been found to be behaving verydifferently from earlier stages of culture. We haveincluded as much of this information as possiblein the descriptions, but these are far fromcomplete and constitute a fascinating area ofinvestigation. In an as yet unpublished studywe subjected a set of cercomonad strains to avariety of culture conditions to see what effectthis had on their morphology and behaviour.In general, the experimental strains did notdiffer radically from the controls (grown in thesame media as for this paper). Therefore,the range of behaviours observed over weeks/months are either ‘normal’ for each strain orprompted by intra- and inter-specific (with bac-teria) interactions in the unnaturally constrainedconditions of 90 mm Petri dishes. Consequently,we suggest that multiple type materials areimportant for cercomonad species (and otherprotists showing similar plasticity) and that animportant element of these should, where possi-ble, include video footage. We have such footage

for all species newly described here, which can bemade available to serious students of the group ifrequired, but we are soon to make them acces-sible on the internet.

Our culturing effort for cercomonads has beenmany fold greater than in any previous studies andwe obtained several fold more clonal cultures thanin all previous studies combined. Nonetheless, wefound only about 22% of previously namedspecies and over four times as many novel ones.This suggests that there are at least hundreds ofcercomonad species and that more intensivestudy could more than quadruple the presentnumber of named species (c. 105). Our unpub-lished environmental data show there are manymore cercomonad lineages than those shown inFigures 14 and 15.

The disparity between culture and rRNA genelibrary sampling has also been observed inglissomonads (Howe et al. 2009) and cercozoanrhizopodial amoebae (Bass et al. 2009a). Massanaet al. (2006) and Jurgens and Massana (2008)suggest that this disparity is caused by theartificial nature of culture environments, oftenspecifically because bacterial concentrations inculture are usually much greater than in theenvironment. It is certainly true that the range ofprotists that appear in cultures with different levelsof added nutrients (and therefore density ofbacteria for grazing) can differ greatly. Morespecific experiments are required to understandthe relationships between bacterial diversity/productivity and protist diversity and grazingactivities/preferences.

The ecological analyses and comparisonsbetween cultured and environmental cercomonaddatasets in this paper are based on the V4 regionalone of the 18S rRNA gene. This initially this isgenerally the fastest evolving region for mosteukaryote groups (Wuyts et al. 2000), but it issometimes identical between strains where otherregions of the 18S (often V2 and V5) differ. Forexample, Paracercomonas spp. Panama107 andPanama61 are identical in the V4 region, but differ inV5. Paracercomonas astra, P. ambulans, and spp.xt159 and a closely related strain (not shown) areeffectively identical in V4 (1 nt difference in xt159),but clearly differ in part of helix 23 outside of the V4region, and in V2, V3, V7, V9, and to a lesser extentin V8. However, this departure from the general ruleis rare in cercomonads, so we used the V4 initially todistinguish between 18S-types. Other regions of thegene were not always present in our data, particu-larly in the environmental clone sequences. There-fore, we have unavoidably underestimated the total

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diversity of cultured strains, and particularly environ-mental genotypes.

Ecological Distribution

The relationship between sets of 18S-types fromculture collections and environmental genelibraries is investigated in more detail in acompanion paper to this one, which also dis-cusses cercomonad ecology and total diversity indetail (Bass et al. 2009b) so these topics do notform part of the present primarily taxonomicpaper. However, some discussion of salinitytolerance is relevant here. Although cercomonadshave been recorded from brackish waters (Mylni-kov and Karpov 2004), we are aware of only oneconfirmed cercomonad strain having been iso-lated from marine or brackish habitats, or fromwater or soils with a constant salinity above 5 %— Paracercomonas marina ATCC50344, isolatedfrom sediment 10-15 m deep in Castle Harbour,Bermuda, but this strain grows preferentially infreshwater media unsupplemented with any salts.Cercomonas marina Mylnikov (1989a), thoughmarine was not a cercomonad but a misidentifiedapusomonad, later renamed Amastigomonas mar-ina Mylnikov (1999). We have confirmed thisreclassification by sequencing the Hsp90 gene ofthe type culture, now dead (Stechmann andCavalier-Smith 2003), which groups with Apuso-monas and not Cercozoa on our unpublishedHsp90 trees. Other Cercomonas species areoccasionally recorded from marine habitats (e.g.Lee and Patterson 2000) but their authenticity astrue cercomonads is often in doubt.

Cercomonad-specific environmental 18S rDNAlibraries (Bass et al. 2007) detected 10 ITS-typesdistributed among three 18S-types in marine(coastal) samples; others are shown in Bass andCavalier-Smith (2004). Salinity tolerance experi-ments reported in Bass et al. (2007) showed thatcercomonad strains vary significantly — evenbetween ITS-types within a single 18S-type — intheir ability to survive and grow in artificial marinemedia (ASW), but with an upper tolerance limit forsurvival (and for some strains only) of half-strengthASW (equivalent to c. 12-20%); many could notgrow even in quarter strength ASW. A.P.M. notesthat any cercomonad strain he has tried to grow ata salinity 45% died. Strains that could grow inquarter to half strength ASW often showed clearmorphological differences from the same cells innormal conditions.

The cercomonad genotypes detected in 18Sand ITS rDNA libraries from coastal marine

samples might represent sub-ITS-type strainsspecifically adapted to more saline conditions, orsimply be cysts that have been passively dis-persed there from the land and cannot grow inthe sea. The abundance and ubiquity of cerco-monads in soil means that cysts must have beenpassing into the sea by the billion for hundreds ofmillions of years; we supect that our failure toisolate any that can live in full strength seawatermeans that none ever succeeded in adapting tosuch conditions. This would agree with thegeneral pattern in protists that the number of taxafound in both marine and non-marine environ-ments is very low (Cavalier-Smith and Nikolaev2008; Cavalier-Smith et al. 2009; Richards andBass 2005). Interestingly, however, P. marina isclosely related to P. minima, which included one ofthe most tolerant strains to ASW media in Bass etal. (2007). Perhaps this clade is ancestrally moretolerant of moderate salinity than most othercercomonads.

Our results suggest that Cercomonas is themost lineage-rich and abundant of the four genera,being represented by 88 independent isolates toParacercomonas’s 63, although P. minima was themost frequently cultured 18S-type overall,accounting for more than a third of the total cultureisolates for Paracercomonas. Eocercomonas andCavernomonas lag far behind with totals of 12 andthree independent isolates respectively.

Our major taxonomic revision of cercomonadscalls into question virtually all previous records ofcercomonads in ecological or faunistic surveys.By far the most common records for soil are forjust two ‘species’ Cercomonas longicauda andParacercomonas (=Cercomonas) crassicauda(Foissner 1991). As neither is the commonest ofthe 59 species studied here in detail over severalyears, we are highly sceptical of all these recordsand consider that excessive lumping into pre-dominantly two meaningless morphospecies haslargely prevailed. Some references to ‘Cercomo-nas crassicauda’ in the literature appear tobe describing a Cercomonas species, not aParacercomonas (e.g. strain C-43 — Cercomonasdactyloptera — Mylnikov (1989b)). In our view onlymolecular means of identification provide theprecision needed for ecological and biogeo-graphic studies of cercomonads.

Cercomonad Morphology

Despite the high levels of variability within strains,some broad correspondences between cercomonad

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phenotypes and phylogeny can be seen. Cerco-monas species are generally larger than those inthe other three genera, usually have proportionallylonger anterior flagella, and have the most meta-bolic cells with often lobed protrusions andpseudopodia. Clade A1b, within Cercomonas,contains the largest cells of all; when more robustphylogenies are available this may merit separa-tion from Cercomonas as a fifth genus. If so wesuggest that it should be named Neocercomonas,in accordance with Ekelund et al. (2004), whoseNeocercomonas is in this clade. Neocercomonascannot be applied to the whole of clade A as cladeA1a contains the type species Cercomonas long-icauda Dujardin, as neotypified in Karpov et al.(2006).

One unexpected discovery is a new genus ofclade A cercomonads, Cavernomonas. Unlike allother cercomonads its surface is rigid — rather asin the apusozoan Amastigomonas — and notsubject to amoeboid movements. From its phylo-genetic position this rigidity is clearly a uniquederived character for this lineage.

A second discovery of broad phylogeneticinterest is that filose branching pseudopodia arewidespread in Eocercomonas and Paracercomo-nas, but (with one exception) absent in the largegenus Cercomonas, which has broader pseudo-pods. From this phylogenetic distribution it wouldappear that the propensity to make branchedfilose pseudopods may be the ancestral characterfor Cercomonadidae and that the broader pseu-dopods of many Cercomonas are a novel specia-lisation. As all genera except Cavernomonas canmake unbranched filose pseudopods (as is truefor most clades in the cercozoan subphylumFilosa) it is very likely that the ancestral cercomo-nad had filose pseudopodia of some kind.

This paper includes the first ultrastructural studyof any Paracercomonas. Its centriolar roots aredramatically simpler than those of either Cerco-monas or Eocercomonas decribed previously(Karpov et al. 2006). Preliminary study of a typicalaerobic Paracercomonas from clade B1 suggeststhat only two slender microtubular roots areassociated with the posterior flagellum, implyingthat this simplicity characterizes the whole genusParacercomonas (Karpov and TC-S unpublished).The roots of monadofilosan outgroups for cerco-monads for which data are available are alsorelatively simple compared to those of clade Acercomonads (Cavalier-Smith et al. 2008, 2009;Karpov et al. 2006). This means that the extremecomplexity of the roots of Cercomonas andEocercomonas is a derived feature for that clade

alone, and is not general for Cercomonadidae.Very likely the ancestral cercomonad had verysimple roots like Paracercomonas. P. anaerobicawas stated by ATCC to be an obligate anaerobe.However, we were able to grow it both anaerobi-cally in totally closed screw-capped tubes com-pletely filled with medium and aerobically insingle-vented Petri dishes. Thus it appears to beonly a facultative anaerobe. This is consistent withour demonstration of mitochondria with normaltubular cristae. The very dense matrix of themitochondria might indicate that they are begin-ning to evolve towards hydrogenosomes, whosematrix is also very dense, but might simply reflectthe generally very high density of the cytoplasm inthis fixation, where the cell may be a littleshrunken. Our demonstration of a ciliary transitionregion proximal hub lattice provides another clade(Cercomonadidae clade B) in which this cer-cozoan synapomorphy (Cavalier-Smith et al.2008) has been found. In contrast to clade Acercomonads, the outer part of this structure doesnot have a dense matrix, so it does not resemble adiaphragm. Distinct lattice fibres are visible,however the circumferential fibre that forms theinner edge of the diaphragm-like structure of cladeA cercomonads is even thicker in P. anaerobica,leaving only a very thin paler space between it andthe central hub. These and the marked differencesin roots from clade A cercomonads furthersupport the separation of Paracercomonas as adistinct genus.

Potential Generic Synonyms

In addition to Dimorpha and Cercobodo (seeResults), Patterson and Zolffel (1991) list thefollowing genera as possible synomyns for Cerco-monas (=cercomonads): Dimastigamoeba Bloch-mann 1894, Prismatomonas Massart 1920, Muk-deniamonas Skvortzow 1960, Changia Skvortzow1960, Reptomonas Kent 1880, Cercomastix Lem-mermann 1913, and Helkesimastix Woodcock andLapage 1914. Based on our experience ofevaluating a wide range of primary sources forthe present paper, we agree that DimastigamoebaBlochmann (1894) is a synonym for Cercomonas(Patterson and Zolffel 1991), as, extremely prob-ably, is Mukdeniamonas. Changia may be acercomonad or pansomonad. However, at leastthree of these genera are not cercomonads.Recently we showed that Helkesimastix doesnot group with cercomonads on 18S rDNA trees,and that its morphology is not concordant withany known cercomonad (Cavalier-Smith et al.

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2009). Prismatomonas appears to be a relative of,or the same as, Helkesimastix, both by virtueof morphology and mode of movement. Repto-monas is probably a Mastigamoeba. Lemmer-mann’s genus Cercomastix and sole speciesC. parva (Hartmann and Chagas, 1910) (Lemmer-mann 1914) was a highly amoeboid anteriorlyuniflagellate saprotroph, 6.5-20mm in length,with a round thick-walled cyst but withoutcontractile vacuoles, and having an elastic axialrod connecting the nucleus to a posterior cerco-monad-like tail. If this rod were really just aposterior flagellum, then this species might be acercomonad, but if not it probably is not; its abilityto swim as well as crawl would also be excep-tional for cercomonads.

Methods

Isolation of cercomonad cultures: Samples were collectedfrom soil and freshwater and marine sediments in sterileEppendorf or Falcon tubes. Samples were hydrated withVolvic water (Danone) or dilutions of the CCAP Artificial SeaWater Medium and left to incubate at room temperature, withor without enrichment (a boiled wheat grain or an aliquot of theenriched water after boiling the grains as a food source forbacteria), for a few days to a few weeks. Depending on theconcentration of organisms present, a 10-100 ml aliquot wasthen serially diluted across eight or twelve wells of 250ml ofenriched Volvic in a 96-well cell culture plate (Nunclon). Theplate was then parafilmed and incubated at room temperaturefor a few days to a couple of weeks. Two or three rounds ofserial dilution were carried out for each isolate, using onlyapparently pure strains to seed the final round. In cases whererDNA sequencing suggested that more than one strain waspresent, further scrutiny of the cultures, and an additionalround of serial dilution were carried out before re-extractingDNA and re-sequencing the strain. Prior to extraction cellswere grown in bulk in 90 mm Petri dishes in enriched Volvic.Most surviving cultures were placed in culture collections: seethe species diagnoses. There were too many cultures for thecollections to accept them all; other cultures are availablefrom the Oxford, Borok, or London (Natural History Museum)laboratories.

Culture experiments: Media were made according toCCAP recipes. Inorganic media were supplemented with asterilized wheat grain to promote bacterial growth. Allexperimental and control (Volvic with grain) cultures weregrown in triplicate in equivalent volumes of medium at roomtemperature in 90 mm Petri dishes (Sterilin, UK). Cultures wereobserved using a x20 dry phase contrast lens on an invertedOlympus IX70 microscope.

Differential interference contrast high definition video-microscopy and electron microscopy: Live cultures werefilmed and photographed using a Nikon Eclipse 80i micro-scope, with a x40 differential interference contrast waterimmersion lens (NA 0.6) and a Sony HDV 1080i Handycams.As far as possible, filming was done 24 hours after inoculationinto enriched Volvic in a glass-bottomed dish. Films wereanalysed on Final Cut Express HD 3.5.1, and digital imageswere exported and transferred to Adobe Photoshop 10.0 CS3

Extended for processing. Other images were made using a40X dry phase contrast lens on an inverted Olympus IX70microscope equipped with a Nikon Coolpix 995 digitalcamera. Some of the Russian strains were imaged separately,on a Biolam-I microscope equipped with a video camera MC-1009/S and phase contrast water immersion 70X objective(KF-5 device). For these, culture characteristics wereobserved on a Panasonic NV-HS850 video recorder; thevideotape recording was then digitized; digital images weretransferred to Photoshop CS3 (Adobe) for processing.Transmission electron microscopy fixation was in glutaralde-hyde followed by osmium tetroxide as detailed previously(Cavalier-Smith et al. 2004). Scanning EM of Cavernomonasmira was carried out as described in Vickerman et al. (2005).

DNA extraction, purification, and sequencing: Environ-mental samples were collected in sterile Eppendorf or Falcontubes. DNA was extracted as soon as possible afterwards byfollowing the Maximum Yield Protocol of the UltraClean SoilDNA Isolation Kit (MoBio Laboratories, CamBio, UK), using0.5-1 g of soil/sediment. For cultures, most of the culturemedium was decanted off, and using a sterile scraper cellswere collected from the bottom of the culture dish, thenconcentrated by centrifugation at 1500 rpm for 15 min at 5 1C.Total DNA was extracted from the pellet using the sameprotocol.

18S rDNA from cultures was PCR-amplified using one ortwo of the following primer sets: 1) 25F and 1256R (Cercozoa-specific: Bass and Cavalier-Smith 2004); 2) 243F and 1733R(Karpov et al. 2006); 3) Cercozoa-specific 1259F and 369R(Karpov et al. 2006). ITS2 rDNA sequences were obtained byamplifying with primer set 4: 1259F and 28Sr1 (50-CGGTACTTGTTCGCTATCGG-30). Environmental DNA wasamplified with primer set 2 or 243F and 1256R (bothcercomonad clade A-specific) for construction of environmentalgene libraries (EGLs); other cercomonad sequences weregenerated from cercozoan-specific EGLs using 25F and1256R as described in Bass and Cavalier-Smith (2004). PCRconditions were: initial denaturation at 95 1C for 5 min, 35 cycles(denaturation at 95 1C for 32 s, annealing at 70 1C (primer sets 1and 3) or 68 1C (primer sets 2, 4, and 243F/1256R) for 36 s,extension at 72 1C for 2.5 min), and final extension at 72 1C for10 min. After sequences of clade A cercomonads were obtainedand verified by phylogenetic analyses, they were checkedagainst the ‘clade A-specific’ primer sequences; the incidenceof mismatch between the 18S and primer sequences are shownon Supplementary Table S1.

PCR products were run on a 1% agarose gel. Bands ofappropriate size were excised, and cleaned following theprotocol of the GFX PCR DNA Gel Band Purification Kit (GEHealthcare, UK). Alternatively, aliquots of the PCR productswere run on a 1% agarose gel and checked for a single bandof correct size. If present, PCR products were cleaned using apolyethylene glycol (PEG) protocol: for a 12.5ml PCR reaction,12.5ml (20%PEG/2.5 M NaCl) PEG was added to each tubeand mixed by vortexing. They were left for 30 min at roomtemperature; then centrifuged at 3000 rpm for 30 min to pelletthe PCR products. Supernatant was discarded by pulse-spinning the inverted tubes at 700 rpm. The pellet was washedwith ice cold 70% ethanol, spun for ten minutes at 3000 rpm,again inverted and pulse-spun to remove the supernatant. Theethanol wash was repeated; the PCR pellet was then re-suspended in de-ionised water, and stored at �20 1C until use.

Directly sequenced culture-derived PCR products mostly gaveunambiguous sequence reads, which were in many casesidentical for multiple isolates from different parts of the world.Therefore intra-individual/clonal variation in cercomonad rDNA

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sequences is likely to be very low, as also shown earlier (Bass etal. 2007). Where such variation occurs it results in ambiguities insequence traces (e.g. Beszteri et al. 2005).

Environmental 18S rDNA libraries were constructed usingthe TOPO TA Cloning Kit (Invitrogen, UK). White colonies werescreened using the original PCR primers and amplicon DNArun on a 1% agarose gel. PCR reaction products thatproduced a band on the gel were cleaned using the PEGprotocol and sequenced using dye terminators and separatedon an automated ABI-377 sequencer. Sequencing primerswere the initial PCR primers and an internal primer Pre3NDf(Karpov et al. 2006). Sequences were edited in BioEdit (Hall1999) and Finch TV (http://www.geospiza.com/finchtv/) andsubmitted to GenBank: accession numbers are given indiagnoses and descriptions, and on Figures 14 and 15.

Sequence analyses: Sequences were aligned in ClustalX(Thompson et al. 1997) and refined by eye in MacGDE (http://www.msu.edu/~lintone/macgde/) or Se-Al (http://tree.bio.e-d.ac.uk/software/seal/). Alignments were masked to omitambiguously aligned positions from the analyses. Somesequences had internal runs of Ns (missing data) resultingfrom the positions of the primers used to amplify them. Thesepositions were also masked out of the analyses for allsequences. Regions including missing data were not usedfor species discrimination (see below) and were generally inconserved regions of the 18S rRNA gene. Putative chimaericsequences (generated by PCR recombination) were identifiedby comparing BioNJ trees constructed by PAUP (Swofford2003) separately for the right and left halves of the alignment.Sequences whose branching position differed between thesetrees were inspected in the alignment and omitted from theanalysis if thought to be chimaeric.

Each dataset shown in Figures 14 and 15 was analysed bytwo methods: i) maximum likelihood (ML) 100 bootstrapreplicates using the RAxML BlackBox web server (GAMMA+Pwith correction for invariant sites) (Stamatakis 2006) and ii) theMPI (parallel) version of MrBayes3.1 (Altekar et al. 2004;Ronquist and Huelsenbeck 2003) run for 5,000,000 genera-tions using GTR+G (4 discrete categories)+invariant sites, withthe covarion model and autocorrelation; parameter valuesestimated from the data. Trees were sampled every 100generations. The tree search used two MCMCMC runs eachwith four chains (default heat parameters). The likelihoodvalues of the two MCMCMC searches were compared tocheck whether they had converged. Trees sampled before thelikelihood plots reached a plateau were discarded (burn-in). Aconsensus of the remaining trees was generated to showposterior probabilities of the branching pattern. The MLbootstrap values were annotated onto the Bayesian treeswhere the same topologies were recovered.

The 18S rDNA V4 region (rather than the whole gene)was used for determining 18S-type boundaries in theenvironmental datasets as 1) it is generally the most variableof the nine variable regions identified in eukaryotic 18S rRNAgenes (Wuyts et al. 2000), 2) all of our sequence reads, bothfrom cultures and environmental libraries, included this regionas a high quality read (not near the end or beginning of thesequence trace), and 3) to reduce the exposure of delimitationdecisions to sequence ambiguities and PCR/sequencing erroralong the whole gene, and to chimaeric sequences. For theenvironmental data, unique 18S-types were defined assequences with 3 or more differences in the V4 region toensure that the 18S-types were more different from each otherthan could be accounted for by the combined effects of intra-genomic variation in rDNA sequence and PCR/sequencingerrors, as described in more detail in Bass et al. (2007).

However, in some cercomonads, different species can haveidentical V4 regions but differences in other variable regions,as reported in the Results and Discussion. Therefore, fordistinguishing between and defining species from cultures,where longer 18S reads were available, the three or morenucleotide differences were distributed among four variableregions of the 18S: V2+V4+V5+V7. Where an 18S-type wasrepresented by sequences from more than one library orculture, single nucleotide differences were taken as sufficientto distinguish between genotypes.

Acknowledgements

DB and AH were supported by a NERC StandardResearch Grant. Many thanks to the MSc Inte-grative Biology class of 2003-4 for assistance withEGL construction, sequencing clones, and stimu-lating discussion. TC-S thanks NERC and theCanadian Institute for Advanced Research Evolu-tionary Biology Program for Fellowship support,NERC for a research grant, and Brian Oates forelectron micrographs of P. anaerobica. APM issupported by The Russian Foundation of BasicResearch (RFBR) grant 08-04-00244. KV thanksNERC and the Leverhulme Trust for support.

Appendix A. Supplementary materials

The online version of this article contains addi-tional supplementary data. Please visit doi:10.1016/j.jbiomech.2004.06.014.

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