ARTICLE IN PRESS

Protist, Vol. 156, 215—224, August 2005

1Correspondinfax 7 812 3289e-mail aak@ak

& 2005 Elsevdoi:10.1016/j

elsevier.de/protis

http://www.Published online date 11 July 2005

ORIGINAL PAPER

18S Ribosomal RNA Gene Sequences ofCochliopodium (Himatismenida) and thePhylogeny of Amoebozoa

Alexander Kudryavtseva,1, Detlef Bernhardb, Martin Schlegelb, Ema E-Y Chaoc, andThomas Cavalier-Smithc

aDepartment of Invertebrate Zoology, Faculty of Biology and Soil Science, Saint-Petersburg State University,Saint-Petersburg, 199034, RussiabLaboratory of Molecular Evolution and Animal Systematics, University of Leipzig, Leipzig, D-04103,GermanycDepartment of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK

Submitted December 24, 2004; Accepted March 28, 2005Monitoring Editor: Michael Melkonian

Cochliopodium is a very distinctive genus of discoid amoebae covered by a dorsal tectum ofcarbohydrate microscales. Its phylogenetic position is unclear, since although sharing many featureswith naked ‘‘gymnamoebae’’, the tectum sets it apart. We sequenced 18S ribosomal RNA genes fromthree Cochliopodium species (minus, spiniferum and Cochliopodium sp., a new species resemblingC. minutum). Phylogenetic analysis shows Cochliopodium as robustly holophyletic and withinAmoebozoa, in full accord with morphological data. Cochliopodium is always one of the basalbranches within Amoebozoa but its precise position is unstable. In Bayesian analysis it is sister toholophyletic Glycostylida, but distance trees mostly place it between Dermamoeba and a possiblyartifactual long-branch cluster including Thecamoeba. These positions are poorly supported andbasal amoebozoan branching ill-resolved, making it unclear whether Discosea (Glycostylida,Himatismenida, Dermamoebida) is holophyletic; however, Thecamoeba seems not specifically relatedto Dermamoeba. We also sequenced the small-subunit rRNA gene of Vannella persistens, whichconstantly grouped with other Vannella species, and two Hartmannella strains. Our trees suggest thatVexilliferidae, Variosea and Hartmannella are polyphyletic, confirming the existence of two verydistinct Hartmannella clades: that comprising H. cantabrigiensis and another divergent species issister to Glaeseria, whilst Hartmannella vermiformis branches more deeply.& 2005 Elsevier GmbH. All rights reserved.

Key words: Amoebozoa; Cochliopodium; Hartmannella; Vannella; molecular phylogeny; rRNA sequences.

g author;70314261.spb.edu (A. Kudryavtsev)

ier GmbH. All rights reserved..protis.2005.03.003

Introduction

The genus Cochliopodium Hertwig et Lesser, 1874comprises lens-shaped amoebae covered by thetectum, a flexible layer of carbohydrate scales,which covers the dorsal surface only of the

ARTICLE IN PRESS216 A. Kudryavtsev et al.

adhering cell. In locomotion these amoebae forma broad peripheral sheet of hyaloplasm surround-ing the central granuloplasm. Their mitochondriahave branching tubular cristae, and Golgi dictyo-somes are well developed. Some species have anelectron-dense body near the dictyosome (so-called Golgi attachment; Yamaoka et al. 1984) thatis probably a MTOC (Kudryavtsev 2004; Kudryavt-sev et al. 2004).

The phylogenetic position of this genus amongunicellular eukaryotes remains unclear. The tec-tum, distinctly visible by light microscopy, wasformerly considered to be a very delicate flexibletest, like that of Microchlamys or Arcella (DeSaedeleer 1934; Hertwig and Lesser 1874; Leidy1879; Penard 1902); therefore, traditional mor-phology-based classification schemes generallyplaced this genus among the arcellinid testatelobose amoebae (Testacealobosia). However, Bo-vee (1979) recognized the similarity of their flatpseudopodia with those of Vannellidae, Hyalodis-cidae and Flabellulidae and accordingly groupedthe family Cochliopodiidae De Saedeleer, 1934with these families as the order Pharopodida.Page (1987) more conservatively left them inTestacealobosia, but because of new insights intothe nature of the tectum (Bark 1973; Nagatani etal. 1981; Yamaoka et al. 1984) created a separateorder Himatismenida for Cochliopodiidae (i.e.Cochliopodium, Gocevia and Paragocevia). Bark(1973) made the radical suggestion that Cochlio-podium may be more closely related to chryso-monads than to amoebae due to the presence ofsurface scales in both taxa. However, theseamoebae have most morphological features ofGymnamoebia sensu Page, 1987, even though thepresence of the tectum sets them apart from other‘‘gymnamoebian’’ taxa. Rogerson and Patterson(2002) treated Cochliopodiidae as ‘‘gymnamoebaeof uncertain affinities’’, unassigned to an order.The recent classification of Amoebozoa proposedby Cavalier-Smith et al. (2004) abandoned bothGymnamoebia and Testacealobosia as formaltaxa and placed the order Himatismenida in thenew class Discosea, together with the ordersGlycostylida (mainly comprising Vannellidae, Vex-illiferidae and Paramoebidae) and Dermamoebida(Thecamoebidae, i.e. Dermamoeba and Theca-moeba).

These contrasting views stem from the lack ofreliable morphological markers to clearly indicatethe affinities of Cochliopodium. Therefore, itsphylogenetic position needs to be tested bysequence data. This paper attempts to fill thegap by estimating the phylogenetic position of this

genus among protozoa using SSU ribosomal RNAgene sequence data. Additionally, new sequencedata from Vannella persistens and two Hartman-nella strains (Hartmannella sp. 2, and one resem-bling H. cantabrigiensis) are analyzed. Includingalso the new thecamoebid sequences from Fahrniet al. (2003) enables us to test the monophyly ofDiscosea for the first time and provide a morecomplete analysis of overall amoebozoan phylo-geny based on 81 sequences.

Results

A Single Branch for all Cochliopodium spp.

In all distance trees (one of them is shown inFig. 1) the three Cochliopodium species formed asingle strongly supported branch (bootstrap98—100%). This result was independent of thealgorithm applied, since in preliminary maximumparsimony and maximum likelihood trees (nogamma correction) using fewer taxa this branchwas also present with strong support (not shown).In the weighted least squares (WLS) distance trees(Fig. 1) Cochliopodium minus was consistentlysister to the other species (C. spiniferum andCochliopodium sp.), but there was no bootstrapsupport for this relative branching order. In aBayesian likelihood analysis (Fig. 2) Cochliopo-dium was also monophyletic with 1.0 posteriorprobability support, but the branching order ofCochliopodium spp. differed from that of thedistance trees, as C. spiniferum was consistentlya sister to C. minus and Cochliopodium sp. with1.0 posterior probability. The Cochliopodiumbranch is rather long — comparable with the longbranches of pelobionts and varioseans (Fig. 1).The distances separating the species of Cochlio-podium were also relatively long. They werecomparable with, for example, the distancesbetween genera Vannella and Platyamoeba withinvannellids, and longer than those between theleptomyxid genera (Fig. 1).

Position of Cochliopodium withinAmoebozoa

During preliminary analyses a number of treesincluding not only Amoebozoa and opisthokontsbut also representatives of the bikonts (Cavalier-Smith 2002, 2003a) were inferred. The Cochliopo-dium branch grouped within Amoebozoa in allthese trees, close to the base of Amoebozoa.The same occurred in trees containing only

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Rhizamoeba sp.Paraflabellula hoguaeParaflabellula reniformis

Leptomyxa reticulataBOLA868AF3

Chaos nobileChaos carolinense

Amoeba proteusAmoeba leningradensis

Hartmannella cantabrigiensisHartmannella sp. 2

Glaeseria miraSaccamoeba limaxHartmannellidae LOS79I

Hartmannella vermiformis ATCC 30966Hartmannella vermiformis CREchinamoeba exundansEchinamoeba thermarum AR1Echinamoeba thermarum GV9Echinamoeba thermarum AG3Echinamoeba thermarum KV2Echinamoeba thermarum AJ489265Echinamoeba thermarum 9Echinamoeba thermarum AJ489264Echinamoeba thermarum MV2

Acanthamoeba tubiashiAcanthamoeba castellaniComandonia operculata

Balamuthia mandrillaris‘Platyamoeba’ stenopodia

Phalansterium ?solitariumMayorella sp.

Pseudoparamoeba pagei CCAP15661Korotnevella stella

Korotnevella monacantholepisA50819Korotnevella hemistylolepisA50804

Neoparamoeba sp.Neoparamoeba sp. C1560

Neoparamoeba sp. A30735Neoparamoeba sp. SM68Neoparamoeba pemaquidensis AFSM3Neoparamoeba pemaquidensis SM53

Vexillifera minutissima

Lingulamoeba leeiClydonella sp. AY183892 Clydonella sp. AY183890

Platyamoeba placidaVannella miroides

Platyamoeba plurinucleolusVannella aberdonicaVannella anglica

Vannella persistensDermamoeba algensis

Thecamoeba similisGephyramoeba sp.

LEMD267AF3Filamoeba nolandi Physarum polycephalum

Didymium nigripesHyperamoeba dachnaya

Stemonitis flavogenitaHyperamoeba sp.50750

Hyperamoeba sp.2 EJCHyperamoeba flagellataDictyostelium discoideum

Mastigamoeba sp.Mastigella commutansPhreatamoeba balamuthiMastigamoeba simplex

Endolimax nanaPelomyxa palustris

Entamoeba gingivalisEntamoeba sp. AF149914

Entamoeba sp. AF149915

Cochliopodium spiniferumCochliopodium sp.

Cochliopodium minus

‘Mastigamoeba invertens’BOLA187AF3

BOLA366AF3

0.10

Vexillifera armata

Centramoebida

Myxogastrea

Phalansteriida

Dictyosteliida

Archamoebae

Conosa

Lobosea

Variosea

Discosea

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Echinamoebidae

Leptomyxoidea

HartmannellidaeEuamoebida

Vannellidae

Breviatea

Vexilliferidae

Paramoebidae

Glycostylida

= 100% support

Himatismenida

Dermamoebida

Varipodida

Mayorellidae

Variosea

Mycetozoa

Dermamoebida

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Figure 1. Weighted least-squares distance tree of 78 amoebozoan and 3 breviate sequences with heuristicsearch. GTR gamma model (i ¼ 0:165745, a ¼ 0:666908). Bootstrapped (128 resamplings). New sequencesshown in bold. Bootstrap support percentages are shown in bold type if over 80%.

Molecular Phylogeny of Cochliopodium 217

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Rhizamoeba sp.Paraflabellula hoguaeParaflabellula reniformis

Leptomyxa reticulataBOLA868 AF372795

Saccamoeba limaxHartmannellidae sp. LOS7N/I

Glaeseria miraHartmannella cantabrigiensisHartmannella sp. 2 (4/3Da/10)

Chaos nobileAmoeba leningradensisChaos carolinense

Amoeba proteusEchinamoeba exundansEchinamoeba thermarum AJ489268Echinamoeba thermarum AJ489262Echinamoeba thermarum AJ489266Echinamoeba thermarum AJ489265Echinamoeba thermarum AJ489267Echinamoeba thermarum AJ489264Echinamoeba thermarum AJ489263Echinamoeba thermarum AJ489261

Hartmannella vermiformis X75514Hartmannella vermiformis AF426157Hartmannella vermiformis X75515Hartmannella vermiformis X75513Hartmannella vermiformis M95168Hartmannella vermiformisAY680840

Mayorella sp.Balamuthia mandrillarisAcanthamoeba tubiashiAcanthamoeba castellaniComandonia operculata

Platyamoeba stenopodiaThecamoeba similis

Dermamoeba algensisCochliopodium spiniferum

Cochliopodium sp.Cochliopodium minus

Lingulamoeba leeiClydonella sp. AY183890

Clydonella sp. AY183892Vannella aberdonicaVannella anglica

Vannella persistensPlatyamoeba plurinucleolusPlatyamoeba placida

Vannella miroidesNeoparamoeba pemaquidensis AY183887Neoparamoeba pemaquidensis AY183889Neoparamoeba pemaquidensis AY183894Neoparamoeba sp. AY193725Neoparamoeba sp. AY193724Neoparamoeba sp. AY193726

Korotnevella hemistylolepisKorotnevella monacantholepis

Korotnevella stellaVexillifera minutissima

Vexillifera armataPseudoparamoeba pagei

Phalansterium ?solitarium

Gephyramoeba sp. Stemonitis flavogenitaHyperamoeba sp. AF093247

Hyperamoeba sp. AF411290Physarum polycephalum

Hyperamoeba flagellataDidymium nigripes

Hyperamoeba dachnaya

LEMD267 AF372778Filamoeba nolandi

Dictyostelium discoideumMastigamoeba sp.

Mastigella commutansPhreatamoeba balamuthiMastigamoeba simplex

Endolimax nanaPelomyxa palustris

Entamoeba gingivalisEntamoeba coli AF149914Entamoeba coli AF149915‘Mastigamoeba invertens’

BOLA187 AF372745BOLA366 AF372746

0.10

Centramoebida

Myxogastrea

Phalansteriida

Dictyosteliida

Archamoebae

Conosa

Variosea

Discosea

Amoebidae

Echinamoebidae

Leptomyxoidea

Hartmannellidae

Euamoebida

Vannellidae

Breviatea

Paramoebidae

Glycostylida

Himatismenida

Varipodida

Mayorellidae

Mycetozoa

Dermamoebida

Hartmannellidae

Vexilliferidae

Variosea

*

**

**

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Lobosea

Protamoebae

Stelamoebea

Acanthamoebidae

Thecamoebidae

Mastigamoebida

Pelobiontida

posterior probability = 1.00

0.92

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Figure 2. Highest likelihood Bayesian tree of 82 Amoebozoa and 3 breviates, using the 8 category gammaplus covarion plus autocorrelation substitution model, 10 million generations and a burn in of 850,000generations. Numbers at nodes are posterior probabilities (in bold if 0.9 or more); they are not shown forCochliopodium and Dermamoeba because their branching order was reversed on the consensus tree; theywere omitted within Echinamoeba thermarum for clarity (they ranged from 0.35 to 0.97). Asterisks indicate theassumed ciliary losses within Amoebozoa. New sequences shown in bold.

218 A. Kudryavtsev et al.

ARTICLE IN PRESSMolecular Phylogeny of Cochliopodium 219

Amoebozoa and opisthokonts (i.e. the unikonts:one of the two major clades of eukaryotes;Cavalier-Smith 2003a; Stechmann and Cavalier-Smith 2003) and breviates. However, the basalbranching within Amoebozoa was ill-resolved; theposition of Cochliopodium varied with taxonsampling and phylogenetic method. In the bestWLS tree with 78 Amoebozoa and 20 opistho-konts as the closest outgroup, plus three breviates(not shown) Cochliopodium branched just withinVannellidae, as sister to the Vannella/Platyamoebaclade, but this was not reproduced even on theconsensus WLS tree with that dataset. Instead, inthe latter tree as well as in the consensus WLStree with the opisthokont outgroup excluded(Fig. 1), the Cochliopodium branch was withinthe apparently paraphyletic Dermamoebida (Der-mamoeba plus Thecamoeba) at the base of aclade comprising Conosa (Archamoebae, Myce-tozoa) and Varipodida (Filamoeba and Gephyra-moeba). This position had only 47% and 30%bootstrap support, respectively. However, therewas strong support for the exclusion of Cochlio-podium from Lobosea sensu stricto in all trees. Asbefore (Cavalier-Smith et al. 2004), the onlystrongly supported bipartition at the base of thetree was between breviates and Amoebozoa.

In view of the partial contradictions amongdistance trees, a Bayesian likelihood analysiswas carried out, using not only gamma correctionbut also intersite correlation and a covarionmodel, as these are likely to model rRNA evolutionmore realistically. On the highest likelihood tree(Fig. 2) Cochliopodium is related to holophyleticGlycostylida, but is their sister rather than branch-ing within Vannellidae, though support is weak. Incontrast, Dermamoebida appear quite stronglypolyphyletic; Dermamoeba is weakly sister toGlycostylida/Himatismenida, whereas Thecamoe-ba is strongly sister to ‘‘Platyamoeba’’ stenopodia,this clade being sister to acanthamoebids withmoderate support. Bayesian analysis also givesstrong support for the exclusion of Mayorella fromParamoebidae and moderate support for itscomplete exclusion from both Glycostylida andDiscosea and a relationship instead to a cladecomprising Acanthamoebidae and Thecamoeba/P. stenopodia.

Phylogenetic Position of the HartmannellaStrains

Two studied strains of Hartmannella robustlygrouped in two different ways. Hartmannella sp.

2 was a fairly distant sister of H. cantabrigiensisAY294147, the two being sisters of Glaeseria withvery strong support (Figs 1—2). By contrast, theCosta Rica strain was essentially identical to theUnited States H. vermiformis strain ATCC 30966(Gunderson et al. 1994), differing only by lackingthe 3 nucleotide ambiguities, the two being sistersto all other Euamoebida except Echinamoeba,but with weak support. In the Bayesian analysis(Fig. 2) the six most divergent available Hartman-nella vermiformis strains were included, confirm-ing the close relationships among them. In none ofthe trees did these two Hartmannella clades grouptogether; bootstrap support for their distinctnesswas very strong at five successive nodes. Othersequenced strains of H. vermiformis differ fromthese at about a dozen variable positions — verymany fewer than those separating them from theH. cantabrigiensis/sp. 2 clade.

Discussion

The Genus Cochliopodium is Monophyletic

Cochliopodium is a rather well-defined morpholo-gically homogeneous genus (Bark 1973; Dykovaet al. 1998; Kudryavtsev 1999, 2000, 2004;Kudryavtsev et al. 2004; Nagatani et al. 1981); itsunique feature, the tectum, is most unlikely tohave been formed several times in evolution. Ourtrees strongly confirm it. The tectal scales of thesequenced species belong to two differenttypes, yet all three robustly form one branch inall trees. Thus Cochliopodium is monophyletic,with the tectum its autapomorphy. The scales ofC. spiniferum and Cochliopodium sp., weaklysisters on distance trees, are of the same type;those of C. minus, which is somewhat moredistant, belong to another. The distance treesuggests early divergence of the two scale types.However, if the more strongly supported Bayesiananalysis is correct, then the spiniferum type isancestral and the minus type secondarily derivedfrom it. The distances separating the Cochliopo-dium species in the trees indicate a high degree ofdiversification among the species. When morespecies are added to the tree it might be reason-able to subdivide them into several genera,especially if they group by scale type.

Cochliopodium Belongs to Amoebozoa

Although the precise position of Cochliopodium inthe phylogenetic tree is unstable, it never moved

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into any protozoan phylum other than Amoebo-zoa. Morphological features of this genus alsoentirely fit the recent circumscription of thisphylum (Cavalier-Smith et al. 2004). Our datacompletely disprove the hypothesis of Bark (1973)that Cochliopodium might be related to somechrysomonad flagellates (kingdom Chromista)based solely on the presence of supposedlysimilar surface scales. However, the chrysomonadscales themselves may not even all be mono-phyletic, as those of Paraphysomonas seem not tobe related to the siliceous scales of synurids andthese two groups are not sisters on the chryso-monad tree (Cavalier-Smith and Chao submitted).

Position of Cochliopodium withinAmoebozoa

The exact position of Cochliopodium withinAmoebozoa cannot be inferred from our data,but several positions can be reasonably con-fidently ruled out. One certain outcome is thefailure of Cochliopodium spp. to group with theclass Lobosea sensu Cavalier-Smith et al. (2004)or the corresponding clade ‘‘Tubulina’’ sensuSmirnov et al. (submitted), which most probablyincludes the testate lobose amoebae (orderArcellinida; Cavalier-Smith et al. 2004). This is inaccordance with morphology, since the lattergroups are characterized by the ability to formcylindrical pseudopodia with monoaxial cytoplas-mic flow, never seen in Cochliopodium. Recentsequence data (Wylezich 2004) confirm thesuggestion that Lobosea also includes the testatelobose amoebae (order Arcellinida). Combinationof these data with the morphological dissimilaritiesbetween Cochliopodium and arcellinids (dis-cussed in e.g. Bark 1973; Ogden 1985; Page1987, 1988) suggests that Cochliopodium spp. arenot testate amoebae, contrary to long-standingtradition.

We have somewhat more confidence in theBayesian tree than in the conflicting distancetrees, since the latter are more likely to suffer fromlong-branch problems and did not use the morerealistic covarion and autocorrelation models andtherefore less accurately represent rRNA evolu-tion. The grouping of Cochliopodium as a sister tothe holophyletic Glycostylida on the Bayesian tree(Fig. 2) corroborates the specific relationship ofthese taxa suggested in figure 8 of Cavalier-Smithet al. (2004). In the Bayesian tree, Dermamoeba issister to a holophyletic Glycostylida plus Cochlio-podium. Both features of the Bayesian tree accord

with the grouping of Himatismenida, Glycostylidaand Dermamoebida as class Discosea (Cavalier-Smith et al. 2004); however their relative branchingorder remains undetermined, as the branchingorder of Dermamoeba and Cochliopodium isreversed on the Bayesian consensus tree, sothere is no support for Cochliopodium beingcloser to glycostylids than is Dermamoeba asFigure 2 shows. Moreover, Thecamoeba, tradi-tionally placed in the family Thecamoebidaetogether with Dermamoeba (Page 1987, 1988),clearly does not group with it on any of our trees.Instead, on the Bayesian tree Thecamoeba groupsstrongly with the problematic ‘‘Platyamoeba’’stenopodia (clearly not a genuine Platyamoeba:Fahrni et al. 2003), and this clade is very distantfrom all other Discosea. All our trees imply thatThecamoebidae and Dermamoebida are not ho-lophyletic; the Bayesian analysis clearly suggeststhat they are polyphyletic; the distance analysesare most simply consistent with polyphyly of theThecamoebidae, but would also allow an inter-pretation of paraphyly. If sequences from otherDermamoeba and Thecamoeba strains confirmthis as well as the grouping of Thecamoeba withthe acanthamoebid/P. stenopodia clade, it will benecessary to place Dermamoeba and Thecamoe-ba in separate families and orders and to excludeThecamoeba from Discosea. Removing Theca-moeba from Dermamoebida and Discosea wouldmake Discosea holophyletic on the Bayesian tree.

On the distance trees Cochliopodium jumpsbetween Glycostylida and Dermamoebida. This isconsistent with the classification of all three ordersas class Discosea (Cavalier-Smith et al. 2004), asit may reflect an affinity of Himatismenida withboth Glycostylida and Dermamoebida. Supportfor all positions found for Cochliopodium is soweak and the base of the tree so poorly resolvedthat one could interpret the results in twocontrasting ways.

Our preferred interpretation is that the Bayesiantree is approximately correct; thus Discoseaminus Thecamoeba is holophyletic and the posi-tion of Cochliopodium on consensus distancetrees is a long-branch artifact. The alternativepossibility that the grouping of both Cochliopo-dium and Dermamoeba with the Varipodida iscorrect would mean that Discosea are paraphy-letic and that their discoid body form wasancestral to the whole clade Discosea/Varipodi-da/Conosa. However, the common ancestor ofsuch a composite clade must have had a cilium(see discussion in Cavalier-Smith et al. 2004),unlike any Discosea so far on the tree. Yet if their

ARTICLE IN PRESSMolecular Phylogeny of Cochliopodium 221

ancestor was ciliated, it seems unlikely that itwould have had the characteristic discoid bodyform and thick, often scaly, surface coat, since noextant ciliated Amoebozoa are discoid with a flatleading pseudopodium: we suspect that a flatanterior pseudopodium and a cilium would befunctionally antagonistic. Therefore, it is probableeither that this discosean body form arose poly-phyletically after separate ciliary losses or morelikely that the distance trees are somewhatmisleading and Discosea (minus Thecamoeba)are really holophyletic, their discoid body formhaving arisen only once after a single commonancestral loss of cilia. Morphological evidencedoes not decisively favor any of these possibilities,but is most consistent with the Bayesian tree.

A third possibility is that all positions of Cochlio-podium on the trees are artifactual, and result fromthe high divergence of its rRNA gene from otherAmoebozoa — it is the fourth longest branch, andunlike the other long branches at present lacks anyclose relatives, which may still be missing from thetree. In this respect, it would be particularlyinteresting to analyze SSU rRNA of Gocevia andParagocevia, included in Himatismenida, due to thepresence of a fibrous cuticle, which, like the tectum,incompletely covers the cell (Page 1987). However,the similarity between tectum and cuticle is rathersuperficial and may be purely convergent, so wetentatively suggest that these genera will probablynot group with Cochliopodium.

Polyphyly and Genetic Diversity ofHartmannella

Our analysis confirms the existence of twobranches within Hartmannella, as found previously(Bolivar et al. 2001; Cavalier-Smith et al. 2004;Fahrni et al. 2003; Smirnov et al., submitted). Onthe basis of those data and dissimilarities betweenH. vermiformis and the other hartmannellids (avery long worm-like locomotive form, more stablepronounced hyaline cap, amphizoic tendenciesand thermotolerance in H. vermiformis) Smirnov etal. (submitted) reasonably suggested that mor-phological features shared by H. vermiformis andother hartmannellids are convergent, but theymight conceivably be ancestral for the apparentclade comprising Hartmannellidae, Amoebidaeand Leptomyxoidea. Our data confirm the deepdivergence of H. vermiformis from the other cladethat is sister to Glaeseria, and show that the latterHartmannella clade is genetically quite diverse.

Our Costa Rica Hartmannella strain differedfrom all previously sequenced European H. vermi-

formis strains but was identical to North Americanstrain ATCC 30966 (originally identified by Page1988), even though its length/breadth ratio wasmore similar to that of H. cantabrigiensis; thusH. vermiformis may be morphologically somewhatmore variable than previously thought. Walochniket al. (2002) also found slight differences inthe cyst of another H. vermiformis strain (C3/8),which is genetically distinct from the other strains(Fig. 2); unfortunately the type strain is notsequenced. The sequence differences amongthe 12 H. vermiformis strains used for the parsi-mony analysis based on the complete sequence(not shown) indicate that this morphospeciesconsists of at least 10 distinctly different ribo-types. Though small compared with those found inmany flagellate morphospecies (Cavalier-Smithand Chao submitted; Von der Heyden andCavalier-Smith 2005; von der Heyden et al.2004a,b), these differences are similar to thoseknown for Acanthamoeba castellani and thoseshown in Figures 1 and 2 for Echinamoebathermarum. It may be the general rule thatamoebozoan morphospecies consist of a numberof ribotypes representing a greater evolutionarydistance than is usual for animal or plant species(typically uniform in 18S rRNA sequence).

General Structure of the Amoebozoan Tree

Our analysis is the most taxonomically completefor Amoebozoa to date, and the first to apply themore realistic covarion and autocorrelation mod-els. Compared with previous trees (Cavalier-Smithet al. 2004, and others cited therein) our Bayesiantree is attractively simple. Setting aside breviates,which, as recent trees suggest, may be moreclosely related to the bikont Apusozoa or Louko-zoa (Cavalier-Smith 2003b, 2004; Cavalier-Smithet al. 2004), there are just three major amoebozo-an clades: two basal clades each clearly ances-trally with cilia and one large derived, entirely non-ciliate clade. If correct, only six losses of cilia needto be invoked (Fig. 2), three within the deepestbranching clade (Archamoebea plus Stelamoe-bea), two within the next clade (Myxogastrea,Varipodida, Phalansteriida) and one only at thebase of the major clade comprising Discosea,Lobosea, Centramoebida, and Mayorella: threefewer losses than in figure 8 of Cavalier-Smithet al. (2004).

Our covarion/gamma/autocorrelation Bayesiananalysis weakly supports suggestions that Con-osa may be paraphyletic (Cavalier-Smithet al. 2004), suggests that Variosea might be

ARTICLE IN PRESS222 A. Kudryavtsev et al.

polyphyletic, and implies that if Phalansteriida andVaripodida were transferred to Conosa, the thuspurified subphylum Protamoebae might be holo-phyletic, not paraphyletic (Cavalier-Smith et al.2004). High support for a clade including Lobosea,Centramoebida, Thecamoeba and Mayorella onour Bayesian tree contrasts markedly with earlierBayesian analysis without a covarion or autocor-relation model, where Lobosea were sister toConosa (Cavalier-Smith et al. 2004). Our treesstrongly indicate that Mayorella lies outsideGlycostylida and may be closer to Centramoebidathan to any Discosea. Its thick surface cuticle(Page 1988) is very different from the surfacescales of Paramoeba and Korotnevella. Thus bothmorphology and the sequence trees indicate thatsuppression of the separate family Mayorellidaeand its inclusion in Paramoebidae (Page 1987,1988) made a polyphyletic group. It is preferableto retain Paramoebidae and Mayorellidae asseparate families (Bovee 1979), so we nowremove Mayorellidae from Glycostylida and Dis-cosea and place it incertae sedis within Prota-moebae (Cavalier-Smith et al. 2004) pendingfurther evidence; this makes Paramoebidae, Gly-

Table 1. Sources and cultivation media of the studied

Species name Source of the str

Cochliopodium spiniferumKudryavtsev, 2004

Bottom sedimentbrackish-water m(Kudryavtsev 200from CCAP (1537

Cochliopodium minus Page,1976

4/1Ab/6D SourhoSusan Brown, NEavailable from CC

Cochliopodium sp. (newspecies, resemblesCochliopodium minutum West,1901)*

Bottom sedimentfreshwater pondGermany

Hartmannella vermiformis** Costa Rica (TC-S

Hartmannella sp. 2 4/3Da/10 SourhoSusan Brown, NE

Vannella persistens CCAP 1589/13 (SBrown 2000)

�This strain is now dead, its total DNA samples and cloA. Kudryavtsev.��This strain now dead had a length/breadth ratio ocantabrigiensis (Page 1988).

costylida and Discosea all more homogeneousmorphologically.

Methods

Cell cultures, DNA isolation, SSU rRNA ampli-fication and sequencing: Three species ofCochliopodium, two strains of Hartmannellaand a strain of Vannella persistens were studied(Table 1). DNA isolation, PCR amplification, clon-ing and sequencing of SSU rRNA genes from thestrains of Hartmannella and V. persistens weredone as described elsewhere (Cavalier-Smithet al. 1995). Total DNA from the cultures ofCochliopodium spp. was isolated using theguanidium thiocyanate method, as described inManiatis et al. (1982). The SSU rRNA gene wasamplified as a single piece by using either theprimer pair 18SFor (forward primer: 50-AAC CTGGTT GAT CCT GCC AGT-30) and 18SRev (reverseprimer: 50-TGA TCC TTC CGC AGG TTC ACCTAC-30) or the primer pair s6 (forward primer:50-CYG CGG TAA TCC CAG CTC-30) and 18SRev.The amplification products were gel-purified and

strains.

ain Cultivation medium

s of aarsh4); available/3)

1.5% NN agar prepared with 6ppt. artificial seawater, feedingon accompanying bacteria

pe soil, UKRC, CEH;AP (1537/5)

1.5% NN agar prepared withPrescott and James (Prescottand James 1955) medium,feeding on Escherichia coli

s of ain Kiel,

1.5% NN agar prepared with PJmedium, feeding on Escherichiacoli

and EEC) ATCC medium 802 with 0.01%cerophyll feeding onaccompanying bacteria

pe soil, UKRC, CEH

1.5% NN agar prepared with PJmedium, feeding on Escherichiacoli

mirnov and 1.5% NN agar prepared with PJmedium, feeding on Escherichiacoli

nes of SSU rRNA gene are available on request from

f only about 4.5, so was initially thought to be H.

ARTICLE IN PRESSMolecular Phylogeny of Cochliopodium 223

cloned in a pGEM-T vector (Promega, USA).Positive clones were sequenced in both directions,using Licor 4200 or ABI 377 sequencers. Completesequences were obtained from the overlappingfragments using Cap Contig Assembly program asimplemented in BioEdit (Tom Hall, USA). Thecomplete SSU rRNA sequence could be obtainedonly from C. spiniferum (GenBank accession no.AY775130). From C. minus and Cochliopodium sp.only the s6/18SRev primer pair gave a product;therefore only partial sequences could be obtained(1481 and 1555 base pairs in length; GenBankaccession nos. AY785056 and AY785057, respec-tively). The other accession numbers are AY680840(H. vermiformis CR), AY68041 (Hartmannella sp. 2strain 4/3Da/10), and AY68042 (V. persistens).

Sequence alignment and phylogenetic ana-lysis: The sequences were aligned manually withour database of over 400 eucaryotic SSU rRNAsequences from GenBank. For inference of phy-logenetic trees a subset of either 78 amoebozoanplus 3 breviates or 78 amoebozoan plus 3 breviateplus 20 opisthokont sequences was selected. Thealignment is available from T. Cavalier-Smith onrequest. 1489 most conserved alignment posi-tions were selected for analysis using PAUP* v.4.0b10 (Swofford 1999) on a Macintosh G4 andG5. Using Modeltest v. 3.06 (Posada and Crandall1998) PAUP* selected the GTR model with gammacorrection for intersite rate variation and allow-ance for invariant sites. Appropriate parameterswere calculated separately for each dataset. Forthe partially incomplete sequences of C. minusand Cochliopodium sp., which had only 1023positions out of 1489 in the whole alignment, aseparate analysis was carried out omitting allmissing positions to ensure that they did notinfluence the tree topology. Distance trees wereinferred using the parameters from Modeltest andthe weighted least-squares method (WLS: power2) with heuristic search; bootstrap analysis used125—128 pseudoreplicates. Separate parsimony(gaps treated as missing data) and BioNJ treeswere calculated for all available strains of H.vermiformis. Mr Bayes v. 3.0b4 was used forBayesian likelihood analysis with the same 1489alignment positions, using the gamma, pluscovarion, plus autocorrelation substitution model.

Acknowlegdements

AK is grateful to Warun Schier for technicalassistance during 5 months research stay at theDepartment of Molecular Evolution and Animal

Systematics of the University of Leipzig, where theCochliopodium sequences were obtained. Furtheranalysis was supported by the Ministry of Educa-tion of Russia (grant no. E02-6.0-78) and RFBR(grant no. 05-04-49000). TCS thanks NERC UK forresearch grants and NERC and the CanadianInstitute for Advanced Research EvolutionaryBiology Programme for Fellowship support.

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