Eukaryotic systematics: a user s guide for cell biologists ... · Eukaryotic systematics: a user’s guide for cell biologists and parasitologists GISELLE WALKER1*, RICHARD G. DORRELL2†,

Eukaryotic systematics a userrsquos guide for cell biologistsand parasitologists

GISELLE WALKER1 RICHARD G DORRELL2dagger ALEXANDER SCHLACHT3dagger

and JOEL B DACKS31Department of Earth Sciences University of Cambridge Downing Street Cambridge CB2 3EQ UK2Department of Biochemistry University of Cambridge Hopkins Building Downing Site Tennis Court Road CambridgeUK CB2 1QW3Department of Cell Biology School of Molecular and Systems Medicine Faculty of Medicine and Dentistry University ofAlberta Edmonton Alberta Canada T6G 2H7

(Received 12 November 2010 revised 22 November 2010 accepted 23 November 2010 first published online 15 February 2011)

SUMMARY

Single-celled parasites like Entamoeba Trypanosoma Phytophthora and Plasmodium wreak untold havoc on human habitatand health Understanding the position of the various protistan pathogens in the larger context of eukaryotic diversityinforms our study of how these parasites operate on a cellular level as well as how they have evolved Here we review theliterature that has brought our understanding of eukaryotic relationships from an idea of parasites as primitive cells to acrystallized view of diversity that encompasses 6 major divisions or supergroups of eukaryotes We provide an updatedtaxonomic scheme (for 2011) based on extensive genomic ultrastructural and phylogenetic evidence with three differinglevels of taxonomic detail for ease of referencing and accessibility (see supplementary material at Cambridge JournalsOn-line) Two of the most pressing issues in cellular evolution the root of the eukaryotic tree and the evolution ofphotosynthesis in complex algae are also discussed along with ideas about what the new generation of genome sequencingtechnologies may contribute to the field of eukaryotic systematicsWe hope that armed with this userrsquos guide cell biologistsand parasitologists will be encouraged about taking an increasingly evolutionary point of view in the battle against parasitesrepresenting real dangers to our livelihoods and lives

Keywords Algae primitive phylogeny parasite

INTRODUCTION

To look around the diversity of life would appear tobe populated by animals plants and fungiAppearances can be deceiving Bacteria aside thevast diversity of cells on the planet today is composedof unicellular eukaryotic microbes or protists Thesepredominantly single-celled creatures can be at thesame time beautiful bizarre and deadly Plasmodiumfalciparum the causative agent of cerebral malariawhich kills close to amillion people a year (Snow et al2005) may be the most notorious protistan pathogenIt is hardly the only one From our top (the brain-eating amoeba Naegleria fowleri) to our tail (gutparasites Giardia and Entamoeba) a myriad array ofmicrobial eukaryotes ndashmay inhabit parasitise andravage the human body Understanding the biologyof these organisms and how they kill is a criticaltask impacting not only human health but global

development as many diseases caused by protistanpathogens have their biggest impacts in AfricaSouth America and South-East Asia (see the WorldHealth Organization webpage for the most recent andmotivating statistics httpwwwwhointvaccine_researchdiseasesen)

While the traditional approach to parasitologyinvolves focused experimental work in the individualorganisms a complementary way forward is to take abroader sweep knitting together genetic medical andcell biological information across a framework ofeukaryotic relationships This allows for comparisonsbetween distantly related parasites yielding insightinto convergently evolved pathogenic mechanisms(eg the regulations of antigenic variation inPlasmodium and Trypanosoma (Duraisingh et al2005 Horn and Barry 2005)) or similarities thatcan be exploited in a single treatment (eg the use ofmetronidazole against anaerobic microbes) It alsoallows for comparisons between the parasites andtheir free-living relatives This can uncover theevolutionary path taken to parasitism and highlightlab-safe model organisms for study (eg the free-living soil amoeba Naegleria gruberi instead of itsdeadly cousin Naegleria fowleri (Fritz-Laylin et al2010)) Finally comparisons amongst parasiticsister-taxa allow for knowledge translation betweenexperimental models and less studied related

To whom correspondence should be addressedDepartment of Cell Biology School of Molecular andSystems Medicine Faculty of Medicine and DentistryUniversity of Alberta Edmonton Alberta Canada T6G2H7 Tel +1-780-248-1493 Fax +1-780-492-0450E-mail dacksualbertaca Department of Anatomy andStructural Biology University of Otago PO Box 56Dunedin 9054 New Zealand E-mails gw265camacukgisellewalkeranatomyotagoacnzdagger Contributed equally

1638

Parasitology (2011) 138 1638ndash1663 copy Cambridge University Press 2011doi101017S0031182010001708

httpsdoiorg101017S0031182010001708Downloaded from httpswwwcambridgeorgcore Open University Library on 20 Jan 2017 at 082337 subject to the Cambridge Core terms of use available at httpswwwcambridgeorgcoreterms

organisms A result in Plasmodium might be appli-cable toEimeria a finding inTrypanosoma bruceimaybe of relevance in Leishmania aetheopicaThis comparative approach means delving into

fields initially appearing tangential to parasitologyOur current understanding of eukaryotic systematicsis based both onmolecular andmicroscopic evidenceConsequently molecular phylogenetics and genomicsare playing an increasingly important roleEvolutionary cell biology and ancient eukaryoticevolution are two of the high-profile fields dependenton eukaryotic systemics They in turn are crucial forthe interpretation of parasitological and cell biologi-cal data in this comparative frameworkIn this review we will discuss the latest literature

on higher-level protist systematics and provide anupdated scheme of the major divisions of eukaryoticdiversity Each of these divisions will be describedhighlighting some of the parasitic organisms and thecurrent and upcoming genome projects Finally wewill explore two of the most pressing controversies ineukaryotic systematics and discuss the anticipatedrole that next generation sequencingmight play in theevolutionary study of parasites and eukaryotes ingeneral

SHIFTING VIEWS ON EUKARYOTIC SYSTEMATICS

FROM lsquoPRIMITIVE rsquo PROTOZOA TO DIVERSE

SUPERGROUPS

The tree of eukaryotes perhaps most familiar to manyparasitologists is that of a collection of crowneukaryotes (animals plants fungi and many algae)with a ladder-like sequential divergence of prominentparasites such as Giardia Trichomonas Encephalito-zoon and Trypanosoma from the base This view ofeukaryotic relationships and the place of parasites init was based on analyses of SSU rDNA genes andsingle protein coding genes performed during theearly to mid-1990s (Sogin 1991 Sogin and Silber-man 1998) Such a phylogeny very much supportedthe Archezoa hypothesis (Cavalier-Smith 19831987) a prevailing paradigm at the time (and stillone sometimes held today) that these ldquoamitochondri-aterdquo protists represent basal eukaryotic lineages thatdiverged before the acquisition of the mitochondrialendosymbiont and other key eukaryotic innovations(eg introns Golgi bodies peroxisomes) If truesuch parasites would be considered primitive andcould be used as possible routes to investigateancestral states of cellular systemsDespite its elegance and logic the Archezoa

hypothesis and the crownbase view of eukaryoticrelationships have been rejected based on several linesof evidence First of all mitochondrially-derivedorganelles (ie hydrogenosomes andmitosomes) havebeen found in nearly all of the proposed ldquoamito-chondriaterdquo organisms (van der Giezen 2009)Initially this was demonstrated by the identifying

of gene sequences of mitochondrial origin (HSP60HSP70 IscU) and later the localizing of these geneproducts (by immuno-microscopy) to double mem-brane-bound organelles found in these taxa (Clarkand Roger 1995 Bui et al 1996 Roger et al 19961998 Tovar et al 2003) Secondly it has been shownthat systematic phylogenetic error such as lsquolongbranch attractionrsquo has had a major effect on thepositioning of these organisms in the tree (Philippeand Germot 2000 Dacks et al 2002) The lessconserved gene sequences were clustered togetherregardless of whether the divergence was due to rapidevolution (as in many parasitic taxa) or a protractedperiod of time in which to accumulate independentmutations (as in the prokaryotic sequences used asoutgroups for the eukaryotic analyses) This artifactcaused otherwise highly divergent protists to bemistaken for basal eukaryotes When accounted forby algorithms andmodels that take differentmodes ofsequence evolution into account (Holder and Lewis2003) the lsquoprimitiversquo parasites and other proposedbasal eukaryotes were either clearly linked withrelatives elsewhere in the tree (as in the case ofMicrosporidia and fungi (Keeling and Fast 2002)) orwere unresolvedThe period following the demise of the Archezoa

hypothesis was one of taxonomic agnosticism andcaution with respect to interpretation of molecularsequence data and the evolution of eukaryotes Suchdata were still of immense value but a growingnumber of researchers abandoned the search for asingle gene that would resolve relationships greatand small in favour of a strategy whereby differentgenes were used to test hypotheses about particulartaxonomic relationships (eg EF2 demonstrating themonophyly of red and green algae (Moreira et al2000) or actin phylogenies uniting the cercozoans andforaminiferans (Keeling 2001)) These resolvedrelationships would then contribute to a consensusview of the eukaryotic tree as a whole It was also atime for a renewed appreciation of ultrastructuraldata and the realization of the need for this to beinterpreted in the light of molecular results (Taylor1999)These ideas crystallized in a seminal paper by

a consortium of taxonomic experts in the diverseprotist organisms An interim taxonomy of eukary-otes was thus provided by Adl et al (2005) based onmorphological ultrastructural and molecular dataand split the tree into six lsquosupergroupsrsquo Opistho-konta Amoebozoa Excavata ArchaeplastidaRhizaria and Chromalveolata By and large thesedivisions have held up and form the basis for thesupergroups that we will describe belowNonetheless work carried out in the 5 years since

the Adl et al paper necessitates changes in thesystematic viewpoint and eventually in the taxo-nomy In some cases the first gene sequences for keytaxa have been obtained shifting their affiliations

1639Eukaryotic systematics


such as the placement of the amoeboid protistFonticula with opisthokonts (Brown et al 2009) orthe placement of various incertae sedis taxa in the tree(eg Stephanopogon with heteroloboseans (Yubukiand Leander 2008))

Moreover the burgeoning availability of genesequences from diverse eukaryotes either throughgenome sequences or from sequence surveys ofexpressed genes have allowed for a new approach toeukaryotic molecular systematics increased use ofmulti-gene concatenated phylogenies Analyses ofgene sequences can not only be affected by systematicartifact but stochastic artifact as well where there issimply insufficient sequence information in a givenmatrix to distinguish between the different possibletree topologies By stringing tens or hundreds ofproteins end to end and treating them as a single datamatrix stochastic error can be reduced This ap-proach has been very powerful in resolving variousissues in eukaryotic systematics providing tremen-dous support for some of the eukaryotic supergroups(Bapteste et al 2002 Rodriguez-Ezpeleta et al 2005Burki et al 2009 Hampl et al 2009) It has alsoallowed a few orphan lineages to find homes such ascentrohelids and telonemids (Burki et al 2009) Acomplementary approach to focusing on extensiveconcatenation is increased taxonomic representationwith fewer genes in the matrix This technique hasconfirmed the validity of many of the supergroupsbut also raised doubts as to others including themonophyly of the Chromalveolata (Parfrey et al2010) the authors are not alone in raising theseconcerns

Originally proposed as an assembly of cryptomo-nads alveolates stramenopiles and haptophytes theChromalveolata account for the overwhelmingmajority of recorded algal species (Cavalier-Smith1999 Simon et al 2009) However new molecularsequence analyses do not support the monophyly ofthese groups to the exclusion of others The issue iswhether two new but well-supported groups con-taining lsquochromalveolatersquo taxa ndash the CCTH clade(Burki et al 2009 Okamoto et al 2009) and SARclade (Cavalier-Smith 2010) ndash should be treated assupergroups Based on their diversity we treat themas such noting that this has no implications fortaxonomic rank

The final advance that has arisen since the Adl et al(Adl et al 2005) taxonomy is in the resolution be-tween the supergroups In two separate concatenatedgene phylogenies resolution was obtained separatingthe excavates amoebozoans and opisthokonts froman assemblage of archaeplastids Rhizaria strameno-piles alveolates and CCTH groupings (Burki et al2008 Hampl et al 2009) Although this resolution isunrooted and thus it is unclear if either of theseassemblages are true clades it still provides an im-portant framework upon which to polarize varioustraits and deduce cellular states of the ancestral

eukaryote It particularly emphasizes that diversemicrobial eukaryotes embedded in the variouseukaryotic supergroups (Table 1) have indepen-dently adopted the parasitic life-style (Fig 1)

EUKARYOTIC SUPERGROUPS

With this historical overview in mind we nowpresent a primer on the diverse and well-supportedmajor eukaryotic divisions (Fig 1) The text here isabbreviated full descriptions of every group andextensive references are provided in the supplemen-tary material (Supplementary material 1 ndash see httpjournalscambridgeorgPAR) The defining featuresof groups are given briefly here these are synapo-morphies only in the cases where they are listed assuch and are less clearly-defined in other cases ndash a lotof systematic research remains to be done Definingfeatures are derived from the published studies citedat the end of each group in the supplementarymaterial We use informal names where possibleand present an indented hierarchy explicitly so as notto imply a formal taxonomic scheme with ranks Thesupplementary section (Supplementary material1 ndash see httpjournalscambridgeorgPAR) explainsthis rationale in detail

We have placed emphasis at the beginning of eachsupergroup description on the synapomorphies thatdefine the group both molecular and ultrastructuralwhere possible We have inset the sub-divisions ineach group again listing the major synapomorphiesand parasitological relevance of example speciesInformation on currently public genome projects isgiven nuclear where possible gene survey ororganelle when this is the best sampling availableHowever due to the fast moving state of these dataand emerging new projects no websites for dataaccess are provided Instead readers are urged tocheck the Genomes Online Database (httpwwwgenomesonlineorg) or the NCBI listing of genomeprojects for the most up to date information

OPISTHOKONTS (James et al 2006 Philippe andTelford 2006Hibbett et al 2007 Shalchian-Tabriziet al 2008)

This supergroup encompasses animals fungi andtheir protistan relatives (Fig 2) Most flagellated taxahave one posteriorly-inserting posteriorly-directedflagellum with a barren second basal body mito-chondrial cristae are flattened There is a synapo-morphic insertion in the EF1-alpha geneOpisthokonts are divided into two principal lineagesholozoa and holomycetes Prominent parasitic taxaexist in both divisions with the parasitic nematodesand Microsporidia being only two of the manyexamples Genome sequencing efforts in this groupare numerous and extensive

1640Giselle Walker and others


Table 1 Where are they now The current eukaryotic affinities of some of the best-known parasites

Name of Disease Host Supergroup Group Subgroup Organism

Schistosomiasis Humans Opisthokonts Metazoa Platyhelminthes Schistosoma mansoniS japonicum

Hookworm Humans Opisthokonts Metazoa Nematodes Ancylostoma duodenaleHydatids Mammals Opisthokonts Metazoa Platyhelminthes Echinococcus speciesFungal pneumonia Humans Opisthokonts Fungi Ascomycetes Pneumocystis cariniiThrush Humans Opisthokonts Fungi Ascomycetes Candida albicansSmut Plants Opisthokonts Fungi Basidiomycetes Ustilago sppRust Plants Opisthokonts Fungi Basidiomycetes Uromyces sppMucormycosis Humans Opisthokonts Fungi Zygomycetes Rhizopus oryzaeZygomycosis Humans Opisthokonts Fungi Zygomycetes Mortierella verticillataChytridiomycosis Amphibians Opisthokonts Fungi Chytrids Batrachochytrium

dendrobatidisNosema Bees Opisthokonts Fungi Microsporidia Nosema apisSilkworm pepperdisease

Silkworms Opisthokonts Fungi Microsporidia Nosema bombycis

Microsporidiosis Humans Opisthokonts Fungi Microsporidia Encephalitozooncuniculi

Microsporidiosis Humans Opisthokonts Fungi Microsporidia Encephalitozoonintestinalis

Amoebic gill disease Salmon Amoebozoa Amoebae Dactylopodids Neoparamoeba sppAmoebic keratitisuveitis encephalitis

Humans Amoebozoa Amoebae Acanthamoebae Acanthamoebacastellanii

Amoebic dysentery Humans Amoebozoa Archamoebae Entamoebae Entamoeba histolyticaBeaver fever giardiasis Humans Excavates Metamonads Diplomonads Giardia lambliaHole in head disease Fish Excavates Metamonads Diplomonads Spironucleus

salmoncidusTrichomoniasis Humans Excavates Metamonads Parabasalia Trichomonas vaginalisPrimary amoebicmeningoencephalitis

Humans Excavates Discoba Heterolobosea Naegleria fowleri

Costia Fish Excavates Discoba Kinetoplastids Ichthyobodo sppCryptobiosis Fish Excavates Discoba Kinetoplastids Cryptobia sppAfrican SleepingSickness

Humans Excavates Discoba Kinetoplastids Trypanosoma brucei

Chagasrsquo disease Humans Excavates Discoba Kinetoplastids Trypanosoma cruziCutaneousleishmaniasis

Humans Excavates Discoba Kinetoplastids Leishmania major

Infantile visceralleishmaniasis

Humans Excavates Discoba Kinetoplastids Leishmania infantum

Kala azar Humans Excavates Discoba Kinetoplastids Leishmania mexicanaProtothecosis Humans Archaeplastids Viridiplantae Trebouxiophytes Prototheca wickerhamiiDodder Plants Archaeplastids Viridiplantae Embryophytes Cuscuta sppEuropean Mistletoe Plants Archaeplastids Viridiplantae Embryophytes Viscum albumBeechdrops Plants Archaeplastids Viridiplantae Embryophytes Epifagus virginianaBlastocystis Humans SAR Stramenopiles Slopalinids Blastocystis hominisSudden oak death Plants SAR Stramenopiles Sloomycetes Phytophthora ramorumPotato blight Plants SAR Stramenopiles Sloomycetes Phytophthora infestansTurf late blight Plants SAR Stramenopiles Labyrinthulids Labyrinthula terrestrisWhite Spot Disease Fish SAR Alveolates Ciliates Ichthyophthirius

multifiliisMalaria Humans SAR Alveolates Apicomplexa Plasmodium

falciparumToxoplasmosis Humans SAR Alveolates Apicomplexa Toxoplasma gondiiEast Coast Fever Bovines SAR Alveolates Apicomplexa Theileria parvaRedwater disease Bovines SAR Alveolates Apicomplexa Babesia bovisCryptosporidiosis Humans SAR Alveolates Apicomplexa Cryptosporidium

parvumDermo Molluscs SAR Alveolates Perkinsids Perkinsus marinusRed Tide na SAR Alveolates Dinoflagellates eg Karenia brevisBrown Tide na SAR Stramenopiles Pelagophytes eg Aureococcus

anophageferrensMSX disease Oysters SAR Rhizaria Haplosporidia Haplosporidium nelsoniPotato powdery scab Plants SAR Rhizaria Phytomyxea Spongospora

subterraneanCabbage club rootdisease

Plants SAR Rhizaria Phytomyxea Plasmodiophorabrassicae



Holozoa Animals choanoflagellates and several re-lated protists Many lineages have unbranched non-tapering tentacles in choanoflagellates and someanimal cells these form a collar surrounding theemergent flagellum There is a conserved gene fusionof ubiquitin and rps30 A close relationship betweenMetazoa and choanoflagellates is well supported

Metazoa (Animals) An extremely diverse groupof multicellular usually motile organisms withextensive cell differentiation diploid except foreggs and sperm with meiosis preceding sexualreproduction Several gene families (eg PRDand ANTP homeobox transcription factors) areuniquely associated with animals Many speciesare parasitic and a few are even known to acquirechloroplasts via kleptoplastidy from algaeGenome sequences include basally divergentlineages (eg the sponge Amphimedon queenslandi-ca the placozoanTrichoplax adhaerens the cnidar-ian Nematostella viridis) and numerous higheranimals (Homo sapiens Drosophila melanogasteribid)

Choanoflagellates Unicellular and colonial collar-flagellates with a single anterior flagellum inside afunnel collar supported by actin filamentsGenome sequences Monosiga brevicollis

Capsaspora owczarzaki Amoebae with very longfilose pseudopodiaCapsaspora is a symbiont of theparasitic snail Biomphalaria glabrata

Ichthyosporids (also Mesomycetozoea) Unicel-lular parasites of vertebrates (dermocystids) and

marine invertebrates (icthyophonids) with a largecentral vacuole and thick cell walls

Holomycetes A diverse clade consisting of fungiand two basally divergent protist lineages (nucleariidsand fonticulids) Pseudopodia where present aretapering and may be branched

Nucleariids Aflagellated amoebae with radiatingfine pseudopodia which consumewhole prey cellsGenome sequences Nuclearia simplex (mitochon-drial only)

Fonticulids Coprophilic cellular slime mouldsMitochondrial cristae are discoidal

Fungi Mycelial and unicellular opisthokontswith chitinous cell walls All studied lineages (exceptfor Microsporidia) synthesize lysine via the α-aminoadipate pathway and uniquely amongsteukaryotes contain genes encoding non-ribosomalpeptide synthetases The phagotrophic microspor-idia and rozellids diverge basally from osmotrophictaxa

Ascomycetes Fungi with a mycelial habit andno flagellated stages Karyogamy meiosis andmembrane division occurs in the ascus a sac-likecell ascospores develop in the cell There is adikaryotic (functionally diploid) mycelium stage inthe life cycle Ascomycetes include symbionts oflichens insects and plants and pathogens orparasites of plants (eg Magnaporthe grisea) andanimals (eg Pneumocystis carinii Candida albi-cans) Genome sequences include Saccharomycescerevisiae Schizosaccharomyces pombe Neurospora

Fig 1 Unrooted tree of eukaryotes This cartoon schematic of eukaryotic diversity shows the classification scheme forthe 6 supergroups and their relative relationships described in the body of this paper The tree is based on the results ofnumerous large-scale genomic and phylogenetic analyses as well as comparative ultrastructural data described withineach text section For complete references see the supplementary materials for each division lsquoJolly Rogerrsquo flags besidetaxonomic groups denote the presence of parasites of agricultural or human importance within that group



crassa Magnaporthe grisea Pneumocystis cariniiand Candida albicans

Basidiomycetes Club fungi mostly with a dikar-yotic mycelial habit and no flagellated stages

Karyogamy and meiosis occur in the basidium acell produced from the mycelium basidiosporesare released and develop exogenously into hyphaeBasidiomycetes include several symbiotic andparasitic taxa (eg the crop pathogen Ustilago

Fig 2 Opisthokonts Panels andashb show holozoans whereas panels cndashg show Holomycetes (a) Metazoan Maluruscyaneus (b) Choanoflagellate Choanoeca sp (c) Nucleariid Nuclearia sp (d) Ascomycete fungus Saccharomycescerevisiae (e) Fungal spore (f) Fungal hyphae (g) Basidiomycete fungus unidentified mushroom species Scale bar ind For a 10 cm e 20 μm f 10 cm g 20 μm Panel a is used with permission from John Walker All other images in thisand subsequent figures are taken from the Microscope website and used under the Creative Commons Licencehttpstarcentralmbledumicroscopeportalphp



maydis and the human pathogen Cryptococcusneoformans) Genome sequences include sapro-trophic (Phanerochaete chrysosporium) ectomycor-rhizal (Laccaria bicolor) and parasitic species(Ustilago maydis Cryptococcus neoformans)

Zygomycetes A paraphyletic array of fungi withlong haploid multinucleate mycelia and noflagellated stages Sexual reproduction occurs viathe fusion of gametangia on the hyphaeZygomycetes include parasites of arthropods andof other fungi and several are potentially lethalhuman pathogens (eg Rhizopus oryzaeMortierella verticillata) Genome sequencesRhizopus oryzae Mortierella verticillata andSmittium culisetae (both mitochondrial only)

Nephridiophagids Parasites of the malpighiantubules of insects with sporous uninucleateamoeboid and multinucleate plasmodial forms

Glomeromycetes Mycorrhizal symbionts ofplants with an asexual life cycle Spores germinateoutside the host on contact with the host myceliadifferentiate into complex tree-like arbuscules withreduced cell walls Genome sequences Glomusintraradices (mitochondrial only)

Chytridiomycetes Coenocytic fungi that formunwalled flagellated zoospores Hydrogenosomesare present in some species Chytridiomycetesinclude parasites of diatoms and amphibians(eg Batrachochytrium dendrobatidis) Genome se-quences Batrachochytrium dendrobatis Monoble-pharella Harpochytrium HyaloraphidiumSpizellomyces and Rhizophydium (all mitochon-drial only)

Blastocladiomycetes Filamentous fungi that formuniflagellate zoospores The nucleus is coveredwith a distinctive cap of ribosomes Species may besaprotrophs or parasites of plants green algaeinvertebrates and other fungi Genome sequencesBlastocladiella emersonii and Allomyces macrogynus(both mitochondrial only)

Microsporidia Amitochondriate aflagellatedintracellular parasites of ciliates and animals (egthe bee parasite Nosema apis the human pathogenEncephalitozoon cuniculi) Non-canonical Golgibodies present Nuclear genomes are the mostcompact of all studied eukaryotes Genome se-quences Encephalitozoon cuniculi and Encephali-tozoon intestinalis

Rozellids Parasites of chytrids blastocladiomy-cetes oomycetes and coleochaete algae withuniflagellate and wall-less amoeboid stagesEncysted and flagellated cells contain conspicuouspolyphosphate granules

Opisthokonts incertae sedis

Corallochytrium Aflagellated marine saprotrophswhich reproduce via multiple rounds of binaryfission and release daughter cells through a pore inthe cell wall

AMOEBOZOA (Page 1987 Shadwick et al 2009)

This supergroup is composed predominantly ofamoebae and amoeboid flagellates (Fig 3) Somespecies have flagella and or subpseudopodia andmany have branching irregular mitochondrial cris-tae The amoebozoa include a number of parasitictaxa (eg Entamoeba histolytica Acanthamoeba cas-tellanii) The supergroup was originally most clearlyidentified from multigene phylogenies with detailsof membership emerging frommore taxon-rich SSUrRNA and actin phylogenies unrooted phylogeniesfrequently support a close relationship betweenamoebozoans and opisthokonts Some traditionalhypotheses of relationships within amoebozoans(eg Lobosea Mycetozoa Conosa Centramoebae)currently have no support as monophyletic cladesand internal relationships are incompletely definedFor ease of retrieval taxa are organized below intoamoebae slime moulds and flagellated amoebae thisis an artificial distinction

lsquoAmoebaersquo

Tubulinea A group of amoebae with diversemorphologies recovered by molecular phylo-geny including Tubulinids Arcellinids andLeptomyxids Pseudopodia are frequently tubularand mono-directional cytoplasmic flow has beenobserved in a wide range of species The hydraparasite Hydramoeba has been proposed to be atubulinid

FlabellinidaDiscosea A group of flattened amoe-bae including Vannellids and Dactylopodids thathave cytoplasm with an anterior hyaline (glassy)zone polydirectional cytoplasmic flow and radiat-ing pseudopodia Lineages within the dactylopo-did genus Neoparamoeba are believed to containperkinsid endoparasites and are themselves associ-ated with amoebic gill disease in farmed salmon

Acanthamoebae Amoebae with clear eruptivepseudopodia at front end of the cell and numerousslender tapering subpseudopodia (acanthopodia)giving the cell a spiny appearance Widely dis-tributed and ecologically dominant in fresh-water and soil habitats acanthamebae containthe human parasite Acanthamoeba castellaniiwhich can cause amoebic keratitis uveitis andencephalitis Genome sequences Acanthamoebacastellanii



Fig 3 Amoebozoa (a) Archamoeba Entamoeba histolytica (b) Archamoeba Mastigamoeba setosa (c) Myxogastridslime mould Polysphondylium spp (d) Myxogastrid slime mould Didymium dachnaya (e) Amoeba Acanthamoebacastellanii (f) Tubulinid amoeba Saccamoeba spp (g) Flabellinid amoeba Vanella spp (h) Thecamoebid amoebaThecamoeba spp Scale bar in a For c 200 μm dndashh 10 μm



Cochliopodids Amoebae with a flexible dorsallayer of trumpet-shaped carbohydrate scales anda distinctive electron-dense body near the Golgiapparatus

Thecamoebae A debated clade of amoebae with athin pellicle and a thick cell coat Many forms arepredators of other amoebae and Sappiniamay be acausative agent of encephalitis

lsquoSlime mouldsrsquo

Dictyostelids Cellular haploid aflagellated filo-podial slime moulds When starved these amoebaecan aggregate in reponse to molecular signalsgenerated by other individuals forming a differ-entiated slug or that fuse to form a zygote whichingests aggregating haploid amoebae Genomesequences Dictyostelium discoideum

Myxogastrids Acellular slime moulds with hap-loid flagellate and filose amoeboid stages whichfuse to form a diploid plasmodium composed ofveins The mitochondrion contains a filamentousnucleoid and branching tubular cristae Genomesequences Physarum polycephalum

lsquoProtostelidsrsquo A collection of groups includingprotosteliids sensu stricto soliformoviidsprotosporangiids cavosteliids ceratiomyxidsand schizoplasmodiids principally acellularwith a fruiting body containing one to fourspores with a cellulose-containing stalk and abasal disc

lsquoFlagellated amoebaersquo

Archamoebae Microareophilic or anaerobic pro-tists with an unusually clear cytoplasm helicalarrays of ribosomes and small non-respiratorymitochondria-like organelles

Entamoebae Amoebae with clear eruptive anteriorpseudopodia andmitosome-like organelles (degen-erate mitochondria) Stacked dictyosomes areabsent Most are intestinal parasites of vertebratesincluding Entamoeba histolytica the causativeagent of amoebic dysentery in humans one is agingival parasite Genome sequences Entamoebahistolytica

Pelobionts Amoeboid flagellates containing asingle basal body connected to a cone and ribbonof microtubules with a distinctive languid or slowflagellar beat One species is an endosymbiont inamphibians Stacked dictyosomes are absentMitochondria-like organelles with metabolismintermediate between hydrogenosomes and mito-somes are present

EXCAVATES (Simpson 2003 Hampl et al 2009Parfrey et al 2010)

The excavates are an assemblage of predominantlyheterotrophic flagellates many of which live inoxygen-poor environments and may contain non-aerobic alternatives to mitochondria (Fig 4) Mostexcavate lineages contain a distinctive longitudinalfeeding groove where suspended food particles arecollected from a current generated by the beating ofposteriorly directed flagella Two major divisionsare currently recognized the principally amito-chondriate metamonads and the predominantlymitochondriate discoba (less formally discobansor occasionally lsquodiscoballsrsquo) Excavates include anumber of major human parasites (eg Trypanosomabrucei Trichomonas vaginalis Giardia intestinalis)Genome sequences are available for the above-mentioned species as well as for several Leishmaniaspecies and for the free-living Naegleria gruberi

Metamonads

Parabasalids Amitochondriate flagellates contain-ing a striated root with attached Golgi dictyosomesthat extends posteriorly from the flagellar appar-atus The feeding groove is secondarily absentParabasalids contain hydrogenosomes degeneratemitochondrially-derived organelles Many speciesare gut commensals of insects (eg Mixotrichaparadoxa hypermastigids) or parasites of ver-tebrates (trichomonads eg Trichomonas vaginalis)Genome sequences Trichomonas vaginalis

Carpediemonads Four flagellate genera ndashCarpediemonas Kipferlia Dysnectes andHicanonectes ndash identified from low-oxygen sedi-ments which resolve paraphyletically at the baseof the fornicates Nomenclature rank and divisionsare not agreed Cells frequently contain acristateorganelles resembling the hydrogenosomes ofparabasalids

Diplomonads+Enteromonads Small amito-chondriate excavate flagellates many of whichhave a doubled cell structure containing twonuclei each attached to a flagellar apparatussupporting a feeding groove There is a mitosomeorganelle homologous to mitochondria and para-basalid hydrogenosomes Stacked dictyosomes areabsent although Golgi homologues have beencharacterized Diplomonads contain several para-sites of humans (eg Giardia lamblia a causativeagent of water-borne enteric disease) and fish(Spironucleus salmoncidus) Genome sequencesGiardia lamblia

Retortamonads Amitochondriate excavates withfour flagella arising from four basal bodies at theanterior end of the feeding groove Stackeddictyosomes are absent The overwhelming



majority of studied species are parasiticChilomastix is a gut commensal of humans Theretortamonads diplomonads + enteromonads andcarpediemonads together form the taxonomicgrouping Fornicata

Oxymonads Amitochondriate flagellates that lackidentifiable Golgi bodies peroxisomes or feedinggrooves and contain a distinctive axostyle made ofmultiple parallel sheets of microtubulesOxymonads are found primarily as symbionts of

Fig 4 Excavates Panels a endashi show discobans whereas Panels bndashd show metamonads (a) Jakobid Reclinomonasamericana (b) Diplomonad Giardia intestinalis (c) Carpediemonas marsupialis (d) Oxymonad Pyrsonympha sp(e) Heterolobosean Percolomonas cosmopolitus (flagellated form) (f) Percolomonas cosmopolitus (amoeboid form)(g) Kinetoplastid Bodo designis (h) Euglenid Euglena mutabilis (i) Kinetoplastid trypanosome Trypanoplasma spScale bar in a For andashc endashg 10 μm i 5 μm



termites or wood-eating cockroaches and mayutilise non-canonical genetic codes

Trimastix Free-living amitochondriate excavatesfour flagella insert orthogonally at the anteriorend of the feeding groove The oxymonads andTrimastix together form the taxonomic groupingPreaxostyla

Discoba

Heterolobosea Free-living heterotrophic exca-vates which contain eruptive pseudopodiaand lack Golgi stacks Some genera (egPsalteriomonas) contain hydrogenosomes andsome mitochondriate species (eg Naegleria gru-beri) may be facultatively anaerobic Naegleriafowleri is an opportunistic pathogen that can infectthe human central nervous system Genomesequences Naegleria gruberi

Jakobids Mitochondriate heterotrophic exca-vates with a sole vane on the dorsal surface of theposterior flagellum The mitochondria of somelineages (eg Reclinomonas americana) retain highlyunreduced genomes and shared genetic featureswith bacteria Genome sequences Reclinomonasamericana (mitochondrial only)

Euglenozoa A diverse group of flagellates in-cluding euglenids kinetoplastids (trypanosomesand bodonids) diplonemids and symbiontidsunified by the presence of a feeding apparatus(cytostome) that may be highly complex and bytwo heterodynamic flagella that contain paraxialrods or lattices The mitochondrial genomesmay be arranged in minicircles (kinetoplasts inkinetoplastids) or small circular chromosomes(diplonemids and euglenids) Predominantly het-erotrophic one lineage of euglenids (includingEuglena gracilis) contains secondary green algal-derived chloroplasts Diplonemids include thefacultative crustacean parasite Rhynchopus kineto-plastids include parasites and endosymbiontsof amoebozoa fish and mammals most notablythe medically important trypanosomes Trypano-soma brucei (causative agent of African SleepingSickness) Trypanosoma cruzi (Chagasrsquo disease)and Leishmania mexicana (kala azar) Genomesequences multiple Trypanosoma species includ-ing Trypanosoma brucei Trypanosoma cruzi mul-tiple Leishmania species including Leishmaniamajor Leishmania infantum and Leishmania brazi-lensis (trypanosomes) Bodo saltans (bodonid inpreparation) Euglena gracilis and Euglena longa(euglenids chloroplasts only)

Excavates incertae sedis

Malawimonads A single genus Malawimonas offree-living mitochondriate excavates containing

an anterior flagellum that inserts apically and aposterior flagellum that inserts at the head of thefeeding groove The mitochondrial genome con-tains bacteria-like features Phylogenetic analysesrecover affinities to both metamonads and discobaGenome sequences Malawimonas jakobiformis(mitochondrial only)

ARCHAEPLASTIDS (Saunders and Hommer-sand 2004 Rodriguez-Ezpeleta et al 2005 Beckerand Marin 2009)

Three phyla Viridiplantae Rhodoplastida andGlaucophyta (Fig 5) that are unified by the presenceof primary plastids believed to have arisen from asingle endosymbiotic event with a cyanobacteriumThis supergroup is also referred to as PlantaeArchaeplastid monophyly is supported by nuclearmultigene phylogenies and discrete features recov-ered in chloroplastids and rhodoplastids (eg acytosolic FBA duplication and the type I transcrip-tion factor pBRp) the branching relationshipsbetween the three constituent phyla are not fullyresolved The archaeplastids include numerous para-sitic taxa both multicellular parasitic plants andunicellular algae Genome sequences have beenproduced for several agriculturally important plants(eg rice grape soya) as well as green algae Therecently published genome of Volvox allowed inves-tigation of another independent example of multi-cellularity Only a single red algal genome is availablebut several are in preparation

Viridiplantae Green algae and plants also referred toas chloroplastids with primary chloroplasts thatcontain thylakoids arranged in stacks and DNAarranged in numerous small nucleoids Uniquelystarch is deposited principally in the chloroplaststroma this has been linked to the conservedduplications of genes involved in starch biosynthesisViridiplantae are divided into chlorophytes andstreptophytes the terms prasinophytes and charo-phytes refer to paraphyletic assemblies within eachclade Four secondary endosymbioses of chloro-phytes by other eukaryotes are known in theeuglenids the chlorarachniophytes the dinoflagellategenus Lepidodinium and the katablepharid Hatenaarenicola

Chlorophytes

Chlorophyceae Haplobiontic chlorophyteswith a transition region in the flagellum consistingof a short proximal and a long distal stellatestructure the latter containing a thick transverseplate structure Genome sequences Chlamy-domonas reinhardtii and Volvox carteri Botry-ococcus braunii and Dunaliella salina (inpreparation)



Fig 5 Archaeplastids Panel a shows a glaucophyte Panels bndashc show red algae Panels dndasho show green plants and algae(a) Glaucophyte Cyanophora paradoxa (b) Rhodophyte Porphyra yezoensis (c) Rhodophyte Porphyridium sp (d)Chlorophyte model organism Chlamydomonas reinhardtii (e) Chlorophyte Eudorina sp (f) Chlorophyte Volvoxcarteri (gndashi) Ulvophyte Wittrockiella sp (j) lsquoPrasinophytersquo Pyramimonas sp (k) lsquoPrasinophyte Nephroselmis olivacea(l) Zygnemophyte desmid Micrasterias sp (m) Streptophyte Mesostigma viride (n) Zygnemophyte desmidClosterium sp (o) Embryophyte tree Acacia melanoxylon Scale bar in a for a c d indashn 5 μm b 5 cm e 20 μm fndashh100 μm o 1m



Ulvophytes Predominantly diplobiontic filamen-tous chlorophytes containing an extremely longtransitional region Species have been identifiedas epibionts on trees red algae and sloth furtrentepohliales are photosymbionts of orangelichen Genome sequences Pseudendocloniumakinetum (mitochondria and chloroplasts) andOltmannsiellopsis viridis (mitochondrial only)

Trebouxiophytes Coccoid and filamentous chlo-rophytes lacking a defined synapomorphy Specieshave been identified as epibionts and endobiontsof plants dinoflagellates marine invertebratesand lichens Two non-photosynthetic generaHelicosporidium and Coccomyxa are parasites ofmarine invertebrates Prototheca is the causativeagent of protothecosis in humans Genome se-quencesChlorella Helicosporidium andPrototheca(mitochondria and chloroplast)

lsquoPrasinophytesrsquo

Chlorodendrales Flagellated prasinophytes cov-ered by an outer layer of stellate and inner layerof diamond-shaped scales The group includesTetraselmis convolutae an acoel endosymbiont

Pycnococcales Flagellated and coccoid prasino-phyes covered by an outer layer of rod-shaped orstellate and inner layer of square or pentagonalscales A member of the pycnococcales is akatablepharid endosymbiont Genome sequencesNephroselmis olivacea (chloroplast only)

Mamiellophytes Very small flagellated or coccoidprasinophytes that may be covered with a spiderweb of flattened scales Genome sequencesOstreococcus tauri Micromonas RCC299

Pyramimonadales Swimming scaly prasinophyteswith four eight or sixteen flagella arising from aninversely pyramidal apical pit Genome sequencesPyramimonas (chloroplast only)

Prasinococcids Naked coccoid prasinophytes con-taining prasinoxanthin mitochondrial membranesintrude into the pyrenoid A sister-group to allother chlorophytes

Streptophytes

Mesostigma viride Asymmetrical unicellular bi-flagellated and filamentous streptophytes coveredwith distinctive maple-leaf shaped scales

Chlorokybus atmophyticus Two- to four-celledsarcinoid packets surrounded by a thick layer ofmucilage lacking plasmodesmata that divide bythe formation of a thin septum With Mesostigma

viride forms the sister-group to all other strepto-phytes Genome sequences chloroplast only

Klebsormidiophytes Charophytes forming un-branched filaments without holdfasts or plasmo-desmata zoospores are released through a pore inthe cell wall

Zygnemophytes Unicellular colonial and un-branched filamentous charophytes lackingplasmodesmata with a cell wall composed ofcrystalline cellulose microfibrils Genome se-quences Spirogyra pratensis (EST only)

Coleochaetales Branched filamentous charo-phytes which bear sheathed hairs zoospores haveunique pyramidal diamond-shaped scales on theflagellum and body Some species may be epi-phytes of charalean algae Genome sequencesColeochaete orbicularis (EST only) Chaetosphaer-idium globosum (mitochondria and chloroplasts)

Charales Charophytes with extremely complexbody structures thalli contain a central axis ofmultinucleate internodal cells and whorls ofbranches radiating from uninucleate node cellsGenome sequences Chara vulgaris (chloroplastonly)

Embryophytes Land plants vascular plantsmosses hornworts and liverworts Cell walls con-tain the complex hemicellulose rhamnogalactur-onan II Over 200 angiosperm genera (egViscum ndashmistletoe Cuscuta ndash dodder Rafflesia)are known to be parasites of other plants Genomesequences many ranging from model angio-sperms (eg Arabidopsis thaliana) to importantcrop species (rice soya) representative gymno-sperms and bryophytes (Pinus taeda Physcomi-trella patens) as well as non-photosyntheticparasites (Epifagus virginiana chloroplast only)

Rhodoplastids Red algae sensu lato also referred to asRhodoplantae Unicellular and multicellular archae-plastids which lack flagella and centrioles at all lifehistory stages chloroplasts contain DNA moleculesarranged in multiple small blebs thylakoids are non-aggregated and embedded with phycobilisomesRhodoplastids utilise a form ID rubisco obtainedfrom a proteobacterial donor Two principle mono-phyletic divisions are known cyanidiophytes andrhodophytes

Cyanidiophytes Unicellular red algae tolerant ofextreme environments with thick proteinaceouscell walls carbohydrates are principally stored asglycogen Genomes are highly reduced and aredepleted of transpososons introns and severalotherwise broadly conserved eukaryotic genefamilies Genome sequences Cyanidioschizonmerolae Galdieria sulphuraria (EST only)



Fig 6 SAR Panels andashh shows stramenopiles indasho show alveolates and pndashx show Rhizaria (a) Stramenochromechrysophyte Dinobryon sp (b) Stramenochrome pedinellid Ciliophrys sp (c) Stramenochrome synuridMallomonas sp (d) Bicosoecid Cafeteria roenbergensis (e) Slabyrinthulid Labyrinthula sp(f) Sloomycete ndash unidentified oomycete (g) Stramenochrome diatom Pleurasigma sp (h) Stramenochromephaeophyte Laminaria digitata (i) Ciliate Aspidisca sp (j) Ciliate Chilodonella sp (k) Ciliate Dileptus sp



Rhodophytes

Rhodellophytes Unicellular rhodophytes with asingle highly lobed plastid surrounded by lipiddroplets storage carbohydrates are predominantlysemi-amylopectins with some amyloses

Porphyridiophytes Unicellular rhodophytes witha single branched or stellate chloroplast lackingan encircling thylakoid in the plastid storagecarbohydrates are principally composed of semi-amylopectins with some amyloses

Stylonematophytes Unicellular pseudofilamen-tous or filamentous rhodophytes cytoplasmicstorage carbohydrates are absent

Compsopogonophytes Rhodophytes with a bi-phasic life history (gametophytes and sporo-phytes) and a central thylakoid-free region ineach chloroplast One order the Erythropeltidalesare principally found as epibionts of marinemacroalgae

Bangiophytes Rhodophytes with a biphasic lifehistory which uniquely produce carposporangiaand spermatangia in distinct packets by successivedivisions Genome sequences Porphyra umbilicalis(in preparation) Porphyra purpurea (EST only)

Florideophytes Branched filamentous rhodo-phytes with a triphasic life history (gametophytescarposporophytes tetrasporophytes) and a dis-tinctive reproductive apparatus consisting ofterminal or lateral carpogonia bearing a longextension for the attachment of spermatangiaSeveral genera (eg Asterocolax HarveyellaHolmsella) are parasites of other closely relatedFlorideophytes

Glaucophytes Small eukaryotes with a plasmamembrane subtended by sacs or shields The blue-green chloroplasts are putatively more primitive thanother primary plastid lineages retaining carboxy-somes (protein-encased bacterial pyrenoids) and apeptidoglycan cell wall No parasitic taxa are knownThree groups are recognised Cyanophorales Glau-cocystales and Gloeochaetales Genome sequencesCyanophora paradoxa (cyanophorale chloroplastonly)

THE SAR CLADE (Leander and Keeling 2003Andersen 2004 Burki et al 2007 Bass et al 2009)

The SAR clade (also referred to as Harosa) is anassembly of the stramenopiles alveolates andRhizaria each of which contain photosyntheticmixotrophic and heterotrophic members (Fig 6)Nuclear multigene phylogenies robustly supportSAR clade monophyly and suggest that the rhizar-ians basally diverge from stramenopiles and alveo-lates Synapomorphies are limited although a novelduplication of the GTPase Rab1 has recently beenidentified in all three phyla The SAR clade containsa number of pathogenic and parasitic genera of majoranthropic interest including Plasmodium (causativeagents of malaria) and Phytophthora (crop patho-gens) Genome sequencing in this group has focusedon parasitic taxa such as the apicomplexans andoomycetes and ecologically prominent ones such asthe diatoms The first sequence of a rhizarian(Bigellowiella natans) is in progress

Stramenopiles A diverse clade of photosynthetic andnon-photosynthetic unicellular and multicellularorganisms Flagella where present are of unevenlength and the long flagellum carries tripartitetubular hairs Several stramenopile taxa are parasitesand pathogens of Metazoa (eg AureococcusBlastocystis) and plants (Phytophthora) Six majorlineages are known currently divided into threemoderately-supported groups labyrinthulomycetesbicosoecids placidids and slopalinids and sloomy-cetes and stramenochromes

Bicosoecids Heterotrophic biflagellates with aningestion area supported by an L-shaped micro-tubular loop Predominantly free-living althoughsome taxa have been identified as chrysophyteepibionts

Labyrinthulids Saprotrophic and heterotrophicstramenopiles with a characteristic secretory orga-nelle (sagenogenetosome) that produces an ecto-plasmic network involved in adhesion and feedingMarine freshwater and terrestrial free-living andepibiotic species are known a parasitic relationshiphas been observed between the species Thrausto-chytrium caudivorum and its flatworm host and thesoil-borne Labyrinthula terrestris has been impli-cated in late blight of turf grass

Placidids Heterotrophic gliding flagellates withtwo unequal flagella containing a double helix inthe transitional region and a distinctive u-shapedmicrotubular root

(l) Apicomplexan Plasmodium vivax (m) Apicomplexan Colpodella vorax (n) Dinoflagellate Peridinium sp(o) Dinoflagellate Oxyrrhis marina (p) Filosan ndash unidentified cercomonad (q) Endomyxan Vampyrella sp(r) Filosan thecofilosan Protaspis tegere (s) Filosan Chlorarachnion reptans (t) Filosan desmothoracid Clathrulinaelegans (u) Filosan Euglypha sp (v) Filosan ndashEbria sp (w) Endomyxan ndash unidentified foraminiferan(x) Radiozoan ndash unidentified acantharean Scale bar in b for andashc e f g i j qndashv 10 μm for d l m p 5 μm h 10 cmw x 100 μm



Sloomycetes Oomycetes sensu lato (hypochytridsoomycetes Developayella) rhizoidal strameno-piles with a flagellated zoospore stage cell wallsgenerally made of cellulose and glycogen andmycolaminarin as storage products A numberof genera (eg Phytophthora AphanomycesSclerophthora) are major biotrophic pathogens ofhigher plants Genome sequences Phytophthorainfestans (the causative agent of potato blight)Phytophthora ramorum (sudden oak death) andPhytophthora sojae (soybean pathogen)

Slopalinids Opalinids sensu lato (opalinids pro-teromonads Blastocystis) flagellated strameno-piles with a ridged cell surface supported bymicrotubular ribbons and a crestal amorphousfibre and with characteristic struts extending fromthe flagellar basal body to the cell surface Somespecies are intestinal commensals of cold-bloodedvertebrates and Blastocystis has been suggested tobe an opportunistic parasite associated with HIVinfection Genome Blastocystis

Stramenochromes Brown algae sensu lato adiverse array of flagellated (eg chrysophytes)coccoid (diatoms) amoeboid (dictyochophytes)and multicellular (phaeophytes) phototrophs andone entirely non-photosynthetic lineage (actino-phryids) Chloroplasts where present contain adistinctive girdle lamella and are surrounded bythree or fourmembranes the outermost of which iscontiguous with the ER Stramenochromes utiliseaureochromes a unique class of blue light receptorBlooms of some marine pelagophytes form harm-ful brown tides some chrysophytes and xantho-phytes are epibiotic or soil-borne symbionts ofplants and at least two diatom lineages have beenuptaken as tertiary chloroplastic endosymbionts bydinoflagellates Genome sequences Aureococcusanophageferrens (pelagophyte) Thalassiosirapseudonana Phaeodactylum tricornutum and Fra-gilariopsis cylindrus (all diatoms) Ectocarpus sili-culosus (phaeophyte) Kryptoperidinium foliaceumand Durinskia baltica (diatom-derived dinoflagel-late endosymbionts chloroplast) Ochromonasdanica (chrysophyte EST only)

Alveolates Predatory phototrophic or parasiticorganisms containing a contiguous layer of corticalalveoli under the cell membrane and an unique familyof associated proteins alveolins Includes sevenlineages that are currently divided into three well-supported groups the ciliates which are basal to allother alveolates apicomplexans colpodellids andchromerids and dinoflagellates perkinsids andellobiopsids Many parasitic taxa are known mostnotably within the Apicomplexa

Ciliates Heterotrophic aerobic and anaerobicalveolates with cilia arranged in lines over the

surface and a complex cell cortex Each cellcontains several small germline nuclei of whichone differentiates to form a large somatic macro-nucleus Free-living in marine freshwater soiland epiphytic environments symbiotic speciesare known eg entodiniomorphids (intestinalcommensalists parasites of mammals) andIchthyophthirius multifiliis (fish parasite) Sometaxa have algal photosymbionts anaerobic speciesmay have methanogenic bacteriosymbiontsGenome sequences Tetrahymena thermophilaParamecium tertauralia Ichthyophthirius multifiliis(EST only)

Apicomplexa Intracellular intestinal or coelomicparasites of metazoa including Plasmodium(causative agent of malaria) Defined by thepresence of an apical complex consisting of a closedconoid a polar ring rhoptries and micronemesinvolved in host cell attachment and invasionApicoplasts non-photosynthetic relict plastidsbound by four membranes and containing agenome with extremely reduced content may bepresent Genome sequences species of Plasmo-dium Toxoplasma (toxoplasmiosis) Theileria(cattle parasite East Coast disease) Babesia (cattleparasite tick fever) and Cryptosporidium (AIDS-associated intestinal parasite)

Colpodellids Free-living predatory flagellateswith hairs or bulbs on the anterior flagellum andan apical feeding complex containing an openconoid which attaches to prey and allows themyzocytotic uptake of cytoplasm

Chromerids Free-living immotile photosyntheticalveolates containing distinctive cone-shapedgolden-brown chloroplasts contacted at the apexby an intracellular cilium The chloroplast containsa circular or long linear genome utilises a form IIrubisco and uniquely amongst photosyntheticalveolates does not contain chlorophyll c Onespecies Chromera velia has been identified aphotosynthetic flagellate CCMP3155 has beenisolated that groups with chromerids and apicom-plexa but the relationships between these lineagesis uncertain Genome sequences Chromera velia(chloroplast only)

Dinoflagellates Phototrophic mixotrophic andheterotrophic alveolates with a coiled transverseflagellum held in a central girdle and a longitudi-nal flagellum in a longitudinal furrow Manyspecies contain a secondary red-algal derivedchloroplast containing peridinin and a form IIrubisco in addition a diverse array of serialchloroplast acquisitions are known The distin-ctive haploid nucleus contains permanently con-densed chromosomes and lacks standard histonesthe genomes of peridinin-containing chloroplastsconsist of multiple small subgenomic minicircles



Some free-living species form harmful red tideblooms others are endobionts of marine invert-ebrates (eg zooxanthellae the primary producersof coral ecosystems) Genome sequencesSymbiodinium spp Alexandrium tamarense (bothEST only)

Perkinsids Intracellular parasites of molluscs anddinoflagellates with a row of bipartite hooks orthick hairs on one side of the anterior flagellum Anapical complex containing an open conoid ananterior and a posterior ring is used to penetratehost cells Perkinsids are non-photosynthetic thereismoderate genetic and ultrastructural evidence forthe retention of plastids Genome sequencesPerkinsus marinus (EST only)

Ellobiopsids Multinucleate parasites principallyof pelagic crustaceans that superficially resemblefungi each individual consists of one or moreexternal tube-like structures and a nutrient-absorbing root and trophic generative structuresinside the host

Rhizaria A major group of eukaryotes with fine root-like reticulate or filose pseudopodia there are nodefining synapomorphies but monophyly is stronglysupported by molecular phylogenies Groups beloware divided into Cercozoa Foraminifera Radiozoaand incertae sedis taxa the relationships between andwithin these groups are incompletely resolvedRhizaria include parasites of algae plants fungi andinvertebrates Multigene studies robustly support aposition for Rhizaria at the base of the SAR cladewhether the Rhizaria historically contained second-ary red algal-derived chloroplast lineages is currentlyunder debate

Cercozoa A diverse assemblage of flagellatesand amoebae that may form filose or reticulatepseudopodia and may harbour endosymbiontscurrently identified on the basis of molecularphylogenies Cercozoa share with Foraminiferaan insertion of one or two amino acids at themonomerndashmonomer junctions of polyubiquitinCercozoa are currently divided into Filosa andEndomyxa

Filosa

Cercomonads Flagellates that produce filosefinger-shaped and branching pseudopodia theanterior flagellum beats stiffly in a cone shape andthe posterior flagellum trails behind the cell

Chlorarachniophytes Reticulate amoebae flagel-lates and or individual filose amoebae with asecondary green algal-derived chloroplast thatretains a highly reduced nucleomorph (relict algalnucleus) Genome sequences Bigelowiella natans

(chloroplast and nucleomorph nuclear in prep-aration)

Clautriavia and Auranticordis Large multi-lobedtetraflagellates (Auranticordis) or small glidinguniflagellates (Clautriavia) with a cell surfacebearing pores and muciferous bodies A quad-riverberis has been reported to contain photo-synthetic endosymbionts which may be ofcyanobacterial origin

Desmothoracids (=Clathrulinids) Heliozoan pro-tists where the cell body is surrounded by axopodiathat protrude through a perforated capsule made ofsilica and organic matter

Euglyphids Filose amoebae with a test of reg-ularly-shaped siliceous plates held together byorganic cement One member Paulinella chroma-tophora has endosymbiotic cyanobacterial-derived cyanelles unrelated to archaeplastidchloroplasts Genome sequences Paulinella chro-matophora (two strains cyanelles only)

Glissomonads Small flagellates that glide on atrailing posterior flagellum and have a shortwaving anterior flagellum

Limnofila Predominantly amoeboid with very finefilose pseudopodia bearing granules (extrusomes)most lineages contain two flagellar stubs that stopat the transitional region

Massisteria Small irregular amoebae from whichradiate thin pseudopodia bearing extrusomes thepseudopodia may branch and anastomose Thereare two flagella which are normally inactive

Metopion Small disc-shaped biflagellates with avery shallow ventral groove at the anterior end andlong posterior and in some taxa stumpy anteriortrailing flagella

Metromonas Small lozenge-shaped gliding flagel-lates with a long posterior and a stumpy anteriortrailing flagellum The posterior flagellum canform a hook shape and attach to the substrate

Pansomonads Heterotrophs with alternating se-dentary amoeboid and motile biflagellate stagesFlagella are hairy heterodynamic and free from thecell body

Sainouroids Small lozenge-shaped gliding fla-gellates with a long posterior and a stumpy anteriortrailing flagellum

Thaumatomonads Rounded heterotrophic flagel-lates with a short anterior scaly flagellum a longnaked posterior flagellum and the ability toproduce filose pseudopodia Many species arecovered in siliceous scales or spines One speciescontains bacterial endosymbionts



Thecofilosea Filose amoebae cells are surroundedby an organic flexible tectum or rigid test (cover-ings) with one or two apertures for filopodia

Cryothecomonas Oval-shaped biflagellates cov-ered with a delicate theca with a pronouncedventral groove from which pseudopodia emergeCryothecomonas is an intracellular parasite ofdiatoms

Ebriids Large marine flagellates with a promi-nent basket-shaped internal siliceous skeleton

Phaeodarea Radiolarian or heliozoan deep-seaprotists with an elaborately decorated skeletonand needles of amorphous silica mixed withorganic and inorganic components The lifecycle is complex and includes flagellated stages

Protaspis Marine flagellates shaped likeelongated ovals with parallel lateral sides withtwo heterodynamic flagella emerging throughfunnels

Pseudodifflugia Filose amoebae with a rigidagglutinated test There may be up to threenuclei per cell

Endomyxa

Ascetosporea Arthropod parasites that producesimple spores

Haplosporidia Invertebrate parasites or hyper-parasites which include pathogens of significantcommercial importance (eg Haplosporidiumnelsoni agent of MSX disease in oysters) Mosttaxa have a distinctive open spore case

Paramyxids Economically important parasitesof bivalve molluscs (eg Marteilia parasite ofSydney rock oysters) crustaceans and annelidswhich make multicellular spores by endogenousbudding

Paradinids Marine parasites of crustaceans (egParadinium a parasite of spot prawns) whichare usually seen as large bag-like spores with aridged surface

Filoreta Amoebae that form extensive multinucle-ate reticulate plasmodial networks Cells are con-nected by cytoplasmic strands that vary from veryfine projections to sheet-like expanses and lackprominent granules

Gromia Large marine rhizopods with filose non-granular pseudopodia and a large ovoid protein-aceous test There is a motile stage with twoflagella

Phytomyxids Plasmodiophorids and phagomyx-ids parasites of vascular plant roots and strame-nopiles which form multinucleate plasmodiabiflagellate zoospores and an invasive attacking

stage that has uniquemodifications of the ER in theintracellular protrusion the stachel and rohr Thegroup includes important agricultural pests egSpongospora subterranea (potato powdery scab) andPlasmodiophora brassicae (cabbage club root dis-ease)

Vampyrellids Large globular multinucleate re-ticulate amoebae with long thick cytoplasmicarms ending in fan-like flat pseudopodia giving riseto filopodia Principally parasites of algae andfungi

Foraminifera Highly diverse marine freshwater andterrestrial rhizopods with large reticulate networksof granular pseudopodia that exhibit unique bidirec-tional rapid cytoplasmic streaming There is alterna-tion of generations and very complex and variablemorphology in the life cycle The group is importantin palaeontology many taxa have chamber-bearingtests used as stratigraphic markers The pseudopodiafrequently contain algal endosymbionts Genomesequences Reticulomyxa filosa (EST only)

Radiozoa Organisms with radiating arms micro-tubule-supported axopodia which extend outwardsfrom a central cell body though a porous organiccapsule to connect with a frothy external layer thatcontains digestive vacuoles and symbionts Theremay be a siliceous or strontium skeleton surroundingthe central cell body Radiozoa lack a polyubiquitininsertion distinguishing them from other rhizarians

Acantharea Radiolarian marine protists withradiating axopodia and spicules of strontiumsulphate The cell is surrounded by a capsule offibrillar material which interconnects myonemesthat control the direction of the spiculesEndosymbiotic algae may be present

Polycystinea Radiolarian marine protists withaxopodia and a siliceous skeleton ranging fromspicules to lattices with radiating spines There is acomplex life cycle including biflagellated swarmersand vegetative colonies The peripheral ectoplasmmay bear symbionts

Sticholonche Kidney-shaped bilaterally sym-metrical marine protists with parallel rows oflocomotory axopodia and rosettes of flattenedsiliceous spicules

THE CCTH CLADE (Smith and Patterson 1986Edvardsen et al 2000 Burki et al 2009 Okamotoet al 2009)

The final major eukaryotic division is the CCTHclade (also named Hacrobia) a group of free-livingheterotrophic mixotrophic and autotrophic organ-isms (Fig 7) Evidence for the group comes fromboth single-gene and multigene phylogenies No



synapomorphies have been reported although simi-lar flagella sub-lamellar vesicles and ejectisomesare observed in most phyla and cryptomonads andhaptophytes share a derived bacterial isoform of thechloroplast-targeted protein rpl36 Cryptomonadspicobiliphytes and kathablepharids are believed to beclosely related there are conflicting data regardingthe phylogenetic positions of haptophytes centro-helids and telonemids No parasitic taxa are knownA genome sequence is available for the haptophyteEmiliania huxleyi a sequence for the cryptomonadGuillardia theta is in preparation

Cryptomonads Mostly autotrophic cells distin-guished by characteristic arrays of bipartite flagel-lar hairs a geometric cell coat and flattenedmitochondrial cristae The secondary red algal-derived chloroplasts retain a nucleomorph (relictalgal nucleus) Cryptomonad-derived plastids havebeen observed in dinoflagellate species these maybe tertiary endosymbionts or may be obtained bykleptoplastidy Genome sequences Guillardiatheta (in progress)

Kathablepharids Free-living heterotrophic flagel-lates with a distinctive spiralled organic sheath

complex conical feeding apparatus and a peripheralER Feeding may occur via engulfment or myzo-cytotic consumption of prey cytoplasm and indi-viduals may swarm to engulf prey Five genera arecurrently known none have true chloroplastsalthough one species Hatena exists with a greenalgal photosymbiont

Picobiliphytes Small (6 μm long) planktonic or-ganisms of unknown general appearance distrib-uted throughout sub-arctic temperate and tropicalwaters described only from environmentalstudies Picobiliphytes have an organelle similarin fluorescence profile to the plastids of red algaeand cryptomonads and have been suggested toretain a nucleomorph

Haptophytes Free-living mixotrophic or auto-trophic flagellates distinguished by a haptonema(a locomotory attachment and feeding organellesupported by microtubules) and distinctive la-mellae in the red algal-derived chloroplasts speciesmay be naked or covered in calcareous organic orin one case siliceous scales Several species (egPhaeocystis globosa) are agents of fish-killing

Fig 7 CCTH (a) Telonemid Telonema subtile (b) Haptophyte Pavlova pinguis (c) Haptophyte coccolithophoridEmiliania huxleyi (d) Cryptophyte Cryptomonas sp (e) Kathablepharid Kathablepharis sp (f) CentrohelidHeterophrys sp Scale bar in f for a 5 μm b 20 μm cndashf 10 μm



planktonic blooms Genome sequences Emilianiahuxleyi Phaeocystis globosa (in progress)

Centrohelids Heterotrophic amoeboid protistswith multiple radiating arms that contain distinc-tive ball-and-cone extrusomes a radiating systemof endocytic vesicles and many Golgi bodiesdispersed through the cell Some species areobserved to form swarms that fuse to form a singlemultinuclear cell during feeding

Telonemids Predatory flagellates with a complexmultilayered lamina and vesicles containing para-crystalline objects beneath the cell surface Onegenus is known but is diverse with a cosmopolitandistribution

EUKARYOTES INCERTAE SEDIS (Foissneret al 1988 Brugerolle et al 2002 Kim et al 2006)

Some eukaryotic taxa remain unplaced throughbeing inadequately described genuinely difficult toplace or genuinely not closely related to any othergroup The taxa listed here are a small proportion ofincertae sedis eukaryotes selected on the basis ofrecent publication and current phylogenetic interest

Apusozoa Small gliding biflagellates with atrailing posterior flagellum two basal bodies anda dorsal organic sheath Apusozoa currently consistof AncyromonasPlanomonas (with a single-layeredsheath and flattened mitochondrial cristae) andapusomonads (with a double-layered sheath andtubular cristae) support for this grouping isincomplete Apusozoa share some ultrastructuraland discrete genomic features with bikonts butpredominantly resolve in molecular analyses asclose relatives of the opisthokonts (Fig 1)

Breviates A group containing only one culturedspecies Breviata anathema an amoeboid flagellatewith filose branching pseudopodia at anterior andposterior ends and a large branching mitochon-drion-like structure without cristae (Fig 1)Breviates have been proposed in some recentmulti-gene analyses to be a sister-taxon to theamoebozoans

Collodictyonids (=Diphylleids) Flagellates with adeep ventral feeding groove and a distinctiveflagellar transition zone containing an electron-dense sleeve around the central microtubules andunusual horseshoe-shaped dictyosomes

Colponema Small kidney-shaped flagellates withsubsurface alveoli and a ventral feeding groovethat can emit fine filopodia and is the region ofphagocytosis Ultrastructure suggests possiblerelationships with the alveolates

Hemimastigophora Multiflagellated protists withdiagonally symmetrical dorsal and ventral

subsurface plates and distinctive concentric ex-trusomes are present Ultrastructure suggests apossible relationship with euglenids

Palpitomonas Heterotrophs with two long flagellaand a single mitochondrion that surrounds theGolgi apparatus Molecular phylogenies and ultra-structural similarities suggest a close relationshipto the archaeplastids or the CCTH clade

For a more detailed treatment of eukaryoticsystematics see the supplementary section (seehttpjournalscambridgeorgPAR)

CURRENT CONTROVERSIES WHERE TO ROOT THE

TREE AND HOW TO CLASSIFY THE ALGAE

Despite the advances in eukaryotic systematics seenin recent years there remain two immediate andexciting areas of controversy Firstly as detailedabove the conceptualization of the eukaryotic treestill held by many parasitologists and cell biologists isof a separation of crown eukaryotes (animals plantsand fungi) and simpleprimitive archaezoan organ-isms such as Giardia Trypanosoma or Trichomonas(Cavalier-Smith 1987 Sogin and Silberman 1998)As is obvious from the new and well-supportedgrouping of eukaryotes into component supergroupsthe various parasites do not group together at the baseof the tree (Fig 1) and yet many authors still operateunder modified versions where their organism ofinterest still represents a primitive eukaryote and thusa living fossil for study (Morrison et al 2007Koopmann et al 2009 Yadav et al 2009) TheArchezoa idea remains simple and intuitive and thusalluring but perhaps one of the major reasons why itis still in currency is the lack of strong alternatehypotheses for the root of eukaryotes Although it ispossible to interpret individual pieces of evidence forthe following rooting hypotheses there is no pre-ponderance of evidence for any of the proposedscenarios making this perhaps the greatest openquestion in eukaryotic cellular evolution todayProposed in 2002 the bikont-unikont rooting was

built on the idea that a bifurcation exists betweenan amoebozoan-opisthokont group (unikonts) andthe rest of eukaryotes (bikonts) (Stechmann andCavalier-Smith 2002) It was presumed that theancestral bikonts possessed two basal bodies and twoflagella whereas ancestral unikonts were presumed toonly possess one of each These two groups were alsothought to undergo different types of flagellartransformation upon cell division The posteriorflagellum became the anterior in the next generationin unikonts while the opposite was proposed as thecase for bikonts Molecular data were also putforward to support this split in the form of a genefusion between dihydrofolate-reductase (DHFR)and thymidylate synthase (TS) (Philippe et al



2000) where the fusion was thought to be exclusiveto bikonts (Stechmann and Cavalier-Smith 2002)as well as the presence of type II myosin onlypresent in unikonts (Richards and Cavalier-Smith2005)

This rooting was dependent on the clean distri-bution of the bikont versus unikont basal body andflagellar arrangement However this was not uni-versally supported by the data available in theliterature even at the time the unikont bikont splitwas proposed (see Supplementary Table 1 ndash httpjournalscambridgeorgPAR) suggesting that moreinformation would be needed before these flagellardata could be regarded as a good heuristic for thisparticular rooting proposal Also the recently pro-posed classifications of the incertae sedis taxa apuso-monads as sister to opisthokonts (Kim et al 2006)and Breviata as sister to amoebozoans (Minge et al2009) call into question the rooting as they eachpossess the lsquowrongrsquo type of flagellar apparatus(Karpov and Zhukov 1986 Walker et al 2006)This rooting hypothesis was also supported by thedistribution of the DHFR-TS fusion (Stechmannand Cavalier-Smith 2002) however the presence inapusomonads of this fusion gene challenges thevalidity of this conclusion

Several more recent rooting hypotheses have beenproposed Based on the distribution pattern of rareconserved amino acids in orthologous genes Rogozinet al (Rogozin et al 2009) deduced that the root ofeukaryotes lies between the archaeplastids and allother taxa This rooting would imply that theacquisition of the cyanobacterial primary endosym-biont sparked the first divergence in the eukaryoticline This rooting hypothesis certainly needs furtherinvestigation and continued inclusion of taxa in suchanalyses

Additionally a root lying between euglenozoansand the rest of eukaryotes (neokaryotes) has beenproposed (Cavalier-Smith 2010) This rootingplaces kinetoplastids at the base of the eukaryotictree due to the nature of their cytochrome biosyn-thesis pathway which uses the cytochrome cc1biogenesis mechanism proteins rather than haemlyase a trait shared with bacteria Phylogenetic treesof the mitochondrial pore TOM and the trypanoso-matid porin VDAC support this root (Pusnik et al2009) This is further suggested by the presence ofthe archaeal Cdc6 replication initiator instead of thecanonical eukaryote DNA replication ORC in trypa-nosomes (Godoy et al 2009) however moreevidence is needed to demonstrate the stability ofsuch a root

Even if the root of eukaryotes was clear con-troversy would remain surrounding the precisephylogenetic position and history of selected parasiticeukaryotes a cause for identity crisis in high-profilepathogens such as Plasmodium Phytophthoraand Blastocystis The nature of the conceptual

overlap between the contentious Chromalveolataand the SAR and CCTH clades has importantimplications for the evolution of complex plastids ineukaryotes

Numerous authors have historically suggestedfrom ultrastructure that different combinations ofcryptomonads stramenopiles haptophytes and al-veolates the only phyla containing red algal-derivedchloroplast lineages might be closely related(Whittaker 1969 Lucas 1970 Cavalier-Smith1981) However the four phyla were first unifiedunder the lsquochromalveolate hypothesisrsquo by Cavalier-Smith (Cavalier-Smith 1999) which suggested thatthe red algal-derived chloroplasts in each lineagearose from a common ancestral secondary endosym-biosis and were secondarily lost in non-photosyn-thetic members of each phylum This hypothesishad been supported by chloroplast gene phylogenieswhich strongly recover support for the monophylyof chromalveolate chloroplasts suggesting that theyoriginated from a single secondary endosymbioticevent (Yoon et al 2004 Li et al 2006 Iida et al2007) In addition there is clear evidence (based onthe retention of diminished non-photosyntheticchloroplasts endosymbiont-derived genes and frombasally divergent photosynthetic lineages) ofsecondary endosymbiont loss in non-photosyntheticalveolates (Moore et al 2008 Reyes-Prieto et al2008 Slamovits and Keeling 2008 Joseph et al2010)

The lsquochromalveolatersquo hypothesis has been chal-lenged on two grounds Firstly recent analyseshave suggested that a much greater number of non-photosynthetic lineages resolve with the lsquochromal-veolatesrsquo than was previously imagined molecularphylogenies strongly support the unification ofcryptomonads and haptophytes with the non-photo-synthetic centrohelids katablepharids and telone-mids in the lsquoCCTH cladersquo (Burki et al 2009Okamoto et al 2009) more surprisingly multigenephylogenies and the conserved presence of a novelRab GTPase have suggested that the primarily non-photosynthetic Rhizaria cluster with the plastid-containing stramenopiles and alveolates in the lsquoSARcladersquo (Burki et al 2007 Elias et al 2009 Cavalier-Smith 2010) As there is no evidence currentlysupporting secondary loss of chloroplasts in theselineages it must be asked whether an ancientendosymbiosis event followed bymultiple secondarylosses is necessarily more parsimonious than theindependent or serial acquisition of chloroplasts byphotosynthetic chromalveolates

Secondly and perhaps more critically it is notknownwhether the CCTH and SAR clades are in factsister-groups or whether the chromalveolates sensulato are paraphyletic while some nuclear multigenephylogenies do support chromalveolate monophyly(Hackett et al 2007 Burki et al 2009 Nozaki et al2009) many recover a closer relationship between the



CCTH clade and the archaeplastids (eg (Burki et al2007 2008 Hampl et al 2009 Reeb et al 2009)) Assuch a number of alternative hypotheses for theorigin of CCTH and SAR clade plastids have beensuggested principally based on the tertiary transferof a recent secondary endosymbiont within andbetween the two lineages (Sanchez Puerta andDelwiche 2008 Bodyl et al 2009 Baurain et al2010)Two questions must be resolved for the taxonomic

validity and status of the lsquochromalveolatesrsquo to bedecided Firstly and most critically is whether thenuclear lineages of the CCTH and SAR clades aresister-taxa This question has already proven to beexceptionally difficult due to both the presumedancient and deep divergence of the archaeplastidsCCTH and SAR clades (Yoon et al 2004 Okamotoand McFadden 2008) which may obscure clearphylogenetic signals Recently Baurain et al (2010)have demonstrated via an ingenious series of statisti-cal analyses that even accounting for branch lengthand divergence date support for the monophyly ofthe mitochondrial and nuclear lineages of cryptomo-nads haptophytes and stramenopiles is substantiallyweaker than might be expected were they mono-phyletic however a test of comparable rigour has notyet been applied to a CCTH+archaeplastid cladeand it is possible that this result may merely beevidence of an extremely weak phylogenetic signal inchromalveolate taxa Secondly if the chromalveolatesare indeed monophyletic it must be determinedwhether non-photosynthetic lsquochromalveolatesrsquo con-tain sufficient clearly algal-derived genes to suggestan ancestral chloroplast history and whether thischloroplast was likely acquired prior to the radiationof the chromalveolates as opposed to independentlyacquired by multiple lineages This has been ren-dered especially complicated by the recent suggestionthat some or all chromalveolate taxa may havepossessed a cryptic green algal-derived secondaryendosymbiont (Frommolt et al 2008 Moustafa et al2009) It is entirely possible that an ancestralsecondary endosymbiosis could have occurred thuscementing the status of the chromalveolates as akingdom that is not retained in any extant chromal-veolatesThe recent developments and challenges to estab-

lished theories concerning the global evolution of theeukaryotes such as the Archaezoa and chromalveo-late hypotheses and the unikonts-bikont root reflecta general trend away from defining phylogeny solelyon discrete synapomorphies such as rare geneticevents (gene fusions and indels) single ultrastructur-al features and endosymbioses Traditional hypoth-eses have been founded on the explicit assumptionthat these traits would not undergo horizontaltransfer convergent evolution or reversion (egStechmann and Cavalier-Smith 2002) but broadertaxonomic sampling and more detailed phylogenetic

studies have frequently drawn evidence to thecontrary such as the validity of a cytosolic duplicationof GAPDH as a chromalveolate-defining synapo-morphy (Fast et al 2001 Obornik et al 2009Takishita et al 2009) Although there is certainly aplace for the use of rare and discrete events in definingphylogeny it seems ever more necessary that these besupported and used in conjunction with large-scalemultigene phylogenetic data

CONCLUSIONS

Recent systematics of protistan parasites andmicrobial eukaryotes in general has revealed theconsiderable complexity in the relationship betweencircumscriptions of taxa based on light microscopyand those incorporating molecular data The futureof this field will be in the continued application ofmolecular phylogenetics based on extensive se-quence datasets with the careful incorporation ofultrastructural and rare genomic event data to high-light possible analytical artifacts and help us under-stand the connection to observations at the lightmicroscopical level and above The technique ofconcatenating hundreds of protein sequences intomega-alignments for analysis has proven exceedinglypowerful and has both clarified contentious issues aswell as served up some real surprises Nonetheless ithas still failed to overcome the bugbear in recon-structing higher-level eukaryotic relationshipsConcatenated analyses are still only partly able tomitigate lsquolong-branch attractionrsquo effects Indeedunchecked these can be exacerbated as randomnoise and may accumulate faster than phylogeneticsignals (Brinkmann et al 2005 Philippe and Telford2006) and there is no agreed or entirely effective wayto mitigate these artifactsOne potential way to address this issue is to include

sequences from organisms whose genes are moreslowly evolving These are often free-living relativesof parasitic taxa (eg chromerid relatives of apicom-plexans Saprolegniales free-living relatives ofparasitic oomycetes) While once this was a hugelylaborious prospect with the advent of next-generation sequencing it will increasingly becomefeasible Already EST-projects can contribute largeamounts of data but in the next few years it should beincreasingly possible for eukaryotic genome projectsto be produced by a few interested laboratories ratherthan being expensive endeavours limited to genomesequencing centres only (eg Diguistini et al 2009Steuernagel et al 2009) The bottleneck then will notbe sequence acquisition but rather the carefulannotation of cellular systems that will need to bedone manually by expert researchers if the projectsare to be more than simple gene cataloguesNonetheless the addition of these slow-evolvinggene sequences should significantly help to resolvesome of the issues pertaining to the lack of resolution



between the supergroups and provide insight intosome of the more group-specific taxonomic issues Ofparticular interest to parasitologists it will also meanthat genomes of the neglected parasitic diseases willbe sequenced organisms that may not have the large-scale global impact as Plasmodium or Entamoeba butare nonetheless deserving of our attention

Next-generation sequencing may not in itselfovercome problems of lateral gene transfer and thusthe potentially artifactual grouping of phylogeneti-cally distant lineages In fact analyses of the genomesand proteomes of model eukaryotes have in somecases suggested that lateral gene transfer especiallybetween endosymbionts and host lineages may beextensive (eg Nosenko and Bhattacharya 2007Moustafa et al 2009 Worden et al 2009) This isparticularly problematic when considering parasitesand nascent endosymbiont lineages where genetransfer events may blur the boundaries betweenhost and symbiont genomes as in the apicomplexansand the well studied arthropod parasite Wolbachia(Huang et al 2004 Dunning Hotopp et al 2007)Already some authors have suggested portmanteaunomenclature for taxa with distinctive and relativelyunreduced symbionts (eg lsquodinotomsrsquo for the diatom-containing dinoflagellates Kryptoperidinium folia-ceum and Durinskia baltica Imanian et al 2010)Although next generation sequencing is unlikely toprecipitate a widespread renaming of eukaryotes toaccommodate endosymbiotic gene transfer it doesraise the broader conceptual question of the extent towhich the eukaryotes may be considered as areductive neatly branching tree

Regardless of the underlying technical and con-ceptual issues next generation sequencing will alsoopen doors for understanding the parasites them-selves as analyses are done comparing gene expressionby deep-sequencing of cDNA from organisms invarious infective conditions (eg Cantacessi et al(2010ab)) Such studies will be particularly insight-ful if paired with gene expression analyses from closefree-living relatives of the parasites giving a finalexample of the utility of eukaryotic systematics toparasitologists As we stand on the cusp of a new eraof genome studies in protozoan parasites we cananticipate exciting new results and new insights intothe evolution of these creatures that demand ourrespect our curiosity and the best of our efforts todefeat

ACKNOWLEDGMENTS

We applaud the initiative that wasMicroScope and is nowEOL We wish to thank David Patterson AlastairSimpson Andrew Roger Mark Field and Chris Howefor general discussion and Bob Andersen David BassMichael Melkonian Charley OrsquoKelly Fred Spiegel andGary Saunders for specific help on aspects of this manu-script as well as general insight Finally we want to thankEmily Herman for help with production of Fig 1

FINANCIAL SUPPORT

This work was supported by fellowships from DarwinCollege Cambridge and the John StanleyGardiner Fund ofthe University of Cambridge and grants from The RoyalSociety and the Templeton Foundation to GW and anNSERC Discovery Grant to JBD

REFERENCES

Adl SM Simpson A G Farmer M A Andersen R AAnderson O R Barta J R Bowser S S Brugerolle GFensome R A Fredericq S James T Y Karpov S Kugrens PKrug J Lane C E Lewis L A Lodge J Lynn D HMann D GMccourt RM Mendoza L Moestrup O Mozley-Standridge S E Nerad T A Shearer C A Smirnov A VSpiegel FW and Taylor M F (2005) The new higher levelclassification of eukaryotes with emphasis on the taxonomy of protistsJournal of Eukaryotic Microbiology 52 399ndash451Andersen R A (2004) Biology and systematics of the heterokont andhaptophyte algae American Journal of Botany 91 1508ndash1522Bapteste E Brinkmann H Lee J A Moore D V Sensen CWGordon P Durufle L Gaasterland T Lopez P Muller M andPhilippe H (2002) The analysis of 100 genes supports the grouping ofthree highly divergent amoebae Dictyostelium Entamoeba andMastigamoeba Proceedings of the National Academy of Sciences USA 991414ndash1419Bass D Chao E E Nikolaev S Yabuki A Ishida K Berney CPakzad U Wylezich C and Cavalier-Smith T (2009) Phylogeny ofnovel naked Filose and Reticulose Cercozoa Granofilosea cl n andProteomyxidea revised Protist 160 75ndash109Baurain D Brinkmann H Petersen J Rodriguez-Ezpeleta NStechmann A Demoulin V Roger A J Burger G Lang B F andPhilippe H (2010) Phylogenomic evidence for separate acquisition ofplastids in cryptophytes haptophytes and stramenopilesMolecular Biologyand Evolution 27 1698ndash1709Becker B and Marin B (2009) Streptophyte algae and the origin ofembryophytes Annals of Botany 103 999ndash1004Bodyl AMackiewicz P and Stiller J W (2009) Early steps in plastidevolution current ideas and controversies Bioessays 31 1219ndash1232Brinkmann H Van Der Giezen M Zhou Y Poncelin DeRaucourt G and Philippe H (2005) An empirical assessment of long-branch attraction artefacts in deep eukaryotic phylogenomics SystematicBiology 54 743ndash757BrownMW Spiegel FW and Silberman J D (2009) Phylogeny ofthe ldquoforgottenrdquo cellular slime mold Fonticula alba reveals a keyevolutionary branch within Opisthokonta Molecular Biology andEvolution 26 2699ndash2709Brugerolle G Bricheux G Philippe H and Coffea G (2002)Collodictyon triciliatum and Diphylleia rotans (=Aulacomonas submarina)form a new family of flagellates (Collodictyonidae) with tubular mitochon-drial cristae that is phylogenetically distant from other flagellate groupsProtist 153 59ndash70Bui E T Bradley P J and Johnson P J (1996) A commonevolutionary origin for mitochondria and hydrogenosomes Proceedings ofthe National Academy of Sciences USA 93 9651ndash9656Burki F Inagaki Y Brate J Archibald JM Keeling P JCavalier-Smith T Sakaguchi M Hashimoto T Horak AKumar S Klaveness D Jakobsen K S Pawlowski J andShalchian-Tabrizi K (2009) Large-scale phylogenomic analyses revealthat two enigmatic protist lineages Telonemia and Centroheliozoa arerelated to photosynthetic chromalveolates Genome Biology and Evolution 1231ndash238Burki F Shalchian-Tabrizi K Minge M Skjaeveland ANikolaev S I Jakobsen K S and Pawlowski J (2007)Phylogenomics reshuffles the eukaryotic supergroups PLoS One 2 e790Burki F Shalchian-Tabrizi K and Pawlowski J (2008)Phylogenomics reveals a new lsquomegagrouprsquo including most photosyntheticeukaryotes Biology Letters 4 366ndash369Cantacessi CMitrevaM Campbell B E Hall R S YoungN DJex A R Ranganathan S and Gasser R B (2010a) First transcrip-tomic analysis of the economically important parasitic nematodeTrichostrongylus colubriformis using a next-generation sequencing approachInfection Genetics and Evolution 10 1199ndash1207Cantacessi C Mitreva M Jex A R Young N D Campbell B EHall R S DoyleM A Ralph S A Rabelo EM Ranganathan SSternberg PW Loukas A and Gasser R B (2010b) Massively



parallel sequencing and analysis of the Necator americanus transcriptomePLoS Neglected Tropical Disease 4 e684Cavalier-Smith T (1981) Eukaryote kingdoms seven or nineBiosystems14 461ndash481Cavalier-Smith T (1983) A 6-kingdom classification and a unifiedphylogeny In Endocytobiology II (eds Schenk H E A and SchwemmlerW) pp 1027ndash1034 Walter de Gruyter BerlinCavalier-Smith T (1987) Eukaryotes with no mitochondriaNature 326332ndash333Cavalier-Smith T (1999) Principles of protein and lipid targeting insecondary symbiogenesis Euglenoid dinoflagellate and sporozoan plastidorigins and the eukaryote family tree Journal of Eukaryotic Microbiology 46347ndash366Cavalier-Smith T (2010) Kingdoms Protozoa and Chromista and theeozoan root of the eukaryotic tree Biology Letters 6 342ndash345Clark C G and Roger A J (1995) Direct evidence for secondary loss ofmitochondria in Entamoeba histolytica Proceedings of the National Academyof Sciences USA 92 6518ndash6521Dacks J B Marinets A Doolittle W F Cavalier-Smith T andLogsdon JM Jr (2002) Analyses of RNA Polymerase II genes fromfree-living protists phylogeny long branch attraction and the eukaryoticbig bang Molecular Biology and Evolution 19 830ndash840Diguistini S Liao N Y Platt D Robertson G Seidel MChan S K Docking T R Birol I Holt R A Hirst MMardis E Marra M A Hamelin R C Bohlmann J Breuil Cand Jones S J (2009) De novo genome sequence assembly of afilamentous fungus using Sanger 454 and Illumina sequence dataGenome Biology 10 R94Dunning Hotopp J C Clark M E Oliveira D C Foster JMFischer P Torres M C Giebel J D Kumar N Ishmael NWang S Ingram J Nene R V Shepard J Tomkins JRichards S Spiro D J Ghedin E Slatko B E Tettelin H andWerren J H (2007) Widespread lateral gene transfer from intracellularbacteria to multicellular eukaryotes Science 317 1753ndash1756Duraisingh M T Voss T S Marty A J Duffy M F Good R TThompson J K Freitas-Junior L H Scherf A Crabb B S andCowman A F (2005) Heterochromatin silencing and locus repositioninglinked to regulation of virulence genes in Plasmodium falciparum Cell 12113ndash24Edvardsen B Eikrem W Green J C Andersen R A Moon-Van-Der-Staay S Y and Medlin L K (2000) Phylogenetic reconstructionsof the Haptophyta inferred from 18S ribosomal DNA sequences andavailable morphological data Phycologia 39 19ndash35Elias M Patron N J and Keeling P J (2009) The RAB familyGTPase Rab1A from Plasmodium falciparum defines a unique paralogshared by chromalveolates and rhizaria Journal of Eukaryotic Microbiology56 348ndash356Fast NM Kissinger J C Roos D S and Keeling P J (2001)Nuclear-encoded plastid-targeted genes suggest a single common origin forapicomplexan and dinoflagellate plastids Molecular Biology and Evolution18 418ndash426Foissner W Blatterer H and Foissner I (1988) TheHemimastigophora (Hemimastix amphikineta nov gen nov spec) a newprotistan phylum from Gondwanan soils European Journal of Protistology23 361ndash383Fritz-Laylin L K Prochnik S E Ginger M L Dacks J BCarpenter M L Field M C Kuo A Paredez A Chapman JPham J Shu S Neupane R Cipriano M Mancuso J Tu HSalamov A Lindquist E Shapiro H Lucas S Grigoriev I VCande W Z Fulton C Rokhsar D S and Dawson S C (2010) Thegenome of Naegleria gruberi illuminates early eukaryotic versatility Cell140 631ndash642Frommolt R Werner S Paulsen H Goss R Wilhelm CZauner S Maier U G Grossman A R Bhattacharya D andLohr M (2008) Ancient recruitment by chromists of green algal genesencoding enzymes for carotenoid biosynthesis Molecular Biology andEvolution 25 2653ndash2667Godoy P D Nogueira-Junior L A Paes L S Cornejo AMartins RM Silber AM Schenkman S and Elias M C(2009) Trypanosome prereplication machinery contains a singlefunctional orc1cdc6 protein which is typical of archaea Eukaryotic Cell8 1592ndash1603Hackett J D Yoon H S Li S Reyes-Prieto A Rummele S Eand Bhattacharya D (2007) Phylogenomic analysis supports themonophyly of cryptophytes and haptophytes and the association of rhizariawith chromalveolates Molecular Biology and Evolution 24 1702ndash1713Hampl V Hug L Leigh JW Dacks J B Lang B FSimpson A G and Roger A J (2009) Phylogenomic analyses support

the monophyly of Excavata and resolve relationships among eukaryoticldquosupergroupsrdquo Proceedings of the National Academy of Sciences USA 1063859ndash3864Hibbett D S BinderM Bischoff J F BlackwellM Cannon P FEriksson O E Huhndorf S James T Kirk PM Lucking RThorsten Lumbsch H Lutzoni F Matheny P BMclaughlin D J Powell M J Redhead S Schoch C LSpatafora J W Stalpers J A Vilgalys R Aime M CAptroot A Bauer R Begerow D Benny G L Castlebury L ACrous PW Dai Y C Gams W Geiser DM Griffith GWGueidan C Hawksworth D L Hestmark G Hosaka KHumber R A Hyde K D Ironside J E Koljalg UKurtzman C P Larsson K H Lichtwardt R Longcore JMiadlikowska J Miller A Moncalvo JM Mozley-Standridge S Oberwinkler F Parmasto E Reeb VRogers J D Roux C Ryvarden L Sampaio J P Schussler ASugiyama J Thorn R G Tibell L Untereiner W A Walker CWang Z Weir A WeissM White MMWinka K Yao Y J andZhang N (2007) A higher-level phylogenetic classification of the FungiMycological Research 111 509ndash547HolderM andLewis P O (2003) Phylogeny estimation traditional andBayesian approaches Nature Reviews Genetics 4 275ndash284Horn D and Barry J D (2005) The central roles of telomeres andsubtelomeres in antigenic variation in African trypanosomes ChromosomeResearch 13 525ndash533Huang JMullapudi N Lancto C A ScottM AbrahamsenM Sand Kissinger J C (2004) Phylogenomic evidence supports pastendosymbiosis intracellular and horizontal gene transfer inCryptosporidium parvum Genome Biology 5 R88Iida K Takishita K Ohshima K and Inagaki Y (2007) Assessingthe monophyly of chlorophyll-c containing plastids by multi-genephylogenies under the unlinked model conditions Molecular Phylogeneticsand Evolution 45 227ndash238Imanian B Pombert J F and Keeling P J (2010) The completeplastid genomes of the two lsquodinotomsrsquo Durinskia baltica andKryptoperidinium foliaceum PLoS One 5 e10711James T Y Kauff F Schoch C L Matheny P B Hofstetter VCox C J Celio G Gueidan C Fraker E Miadlikowska JLumbsch H T Rauhut A Reeb V Arnold A E Amtoft AStajich J E Hosaka K Sung G H Johnson D Orsquorourke BCrockett M Binder M Curtis J M Slot J C Wang ZWilson AW Schussler A Longcore J E Orsquodonnell KMozley-Standridge S Porter D Letcher PM Powell M JTaylor J W White MM Griffith GW Davies D RHumber R A Morton J B Sugiyama J Rossman A YRogers J D Pfister D H Hewitt D Hansen K Hambleton SShoemaker R A Kohlmeyer J Volkmann-Kohlmeyer BSpotts R A Serdani M Crous PW Hughes KWMatsuura K Langer E Langer G Untereiner W ALucking R Budel B Geiser DM Aptroot A Diederich PSchmitt I Schultz M Yahr R Hibbett D S Lutzoni FMclaughlin D J Spatafora JW and Vilgalys R (2006)Reconstructing the early evolution of Fungi using a six-gene phylogenyNature 443 818ndash822Joseph S J Fernandez-Robledo J A Gardner M J El-Sayed NM Kuo C H Schott E J Wang H Kissinger J C andVasta G R (2010) The Alveolate Perkinsus marinus biological insightsfrom EST gene discovery BMC Genomics 11 228Karpov S A and Zhukov B F (1986) Ultrastructure and taxonomicposition ofApusomonas proboscidea AlexeieffArchiv fuumlr Protistenkund 13113ndash26Keeling P J (2001) Foraminifera and cercozoa are related in actinphylogeny two orphans find a home Molecular Biology and Evolution 181551ndash1557Keeling P J and Fast NM (2002) Microsporidia biology andevolution of highly reduced intracellular parasites Annual ReviewsMicrobiology 56 93ndash116Kim E Simpson A G and Graham L E (2006) Evolutionaryrelationships of apusomonads Inferred from taxon-rich analyses of sixnuclear-encoded genes Molecular Biology and Evolution 23 2455ndash2466Koopmann R Muhammad K Perbandt M Betzel C andDuszenko M (2009) Trypanosoma brucei ATG8 structural insights intoautophagic-like mechanisms in protozoa Autophagy 5 1085ndash1091Leander B S and Keeling P J (2003) Morphostasis in alveolateevolution Trends in Ecology and Evolution 18 395ndash402Li S Nosenko T Hackett J D and Bhattacharya D (2006)Phylogenomic analysis identifies red algal genes of endosymbiotic origin inthe chromalveolates Molecular Biology and Evolution 23 663ndash674



Lucas I A N (1970) Observation on the ultrastructure of representativesof the genera Hemiselmis and Chroomonas (Cryptophyceae) BritishPhycological Journal 5 29ndash37Minge M A Silberman J D Orr R J Cavalier-Smith TShalchian-Tabrizi K Burki F Skjaeveland A andJakobsen K S (2009) Evolutionary position of breviate amoebae andthe primary eukaryote divergenceProceedings of the Royal Society of LondonB Biological Sciences 276 597ndash604Moore R B Obornik M Janouskovec J Chrudimsky TVancova M Green DH Wright SW Davies NWBolch C J Heimann K Slapeta J Hoegh-Guldberg OLogsdon JM and Carter D A (2008) A photosynthetic alveolateclosely related to apicomplexan parasites Nature 451 959ndash963Moreira D Le Guyader H and Phillippe H (2000) The origin of redalgae and the evolution of chloroplasts Nature 405 69ndash72Morrison H G Mcarthur A G Gillin F D Aley S BAdam R D Olsen G J Best A A Cande W Z Chen FCipriano M J Davids B J Dawson S C Elmendorf H GHehl A B Holder M E Huse SM Kim U U Lasek-Nesselquist E Manning G Nigam A Nixon J E Palm DPassamaneck N E Prabhu A Reich C I Reiner D SSamuelson J Svard S G and Sogin M L (2007) Genomicminimalism in the early diverging intestinal parasite Giardia lambliaScience 317 1921ndash1926Moustafa A Beszteri B Maier U G Bowler C Valentin K andBhattacharya D (2009) Genomic footprints of a cryptic plastidendosymbiosis in diatoms Science 324 1724ndash1726Nosenko T and Bhattacharya D (2007) Horizontal gene transfer inchromalveolates BMC Evolutionary Biology 7 173Nozaki H Maruyama S Matsuzaki M Nakada T Kato S andMisawa K (2009) Phylogenetic positions of Glaucophyta green plants(Archaeplastida) and Haptophyta (Chromalveolata) as deduced from slowlyevolving nuclear genes Molecular Phylogenetics and Evolution 53 872ndash880Obornik M Janouskovec J Chrudimsky T and Lukes J (2009)Evolution of the apicoplast and its hosts from heterotrophy to autotrophyand back again International Journal for Parasitology 39 1ndash12Okamoto N Chantangsi C Horak A Leander B S andKeeling P J (2009) Molecular phylogeny and description of the novelkatablepharid Roombia truncata gen et sp nov and establishment of theHacrobia taxon nov PLoS One 4 e7080Okamoto N and Mcfadden G I (2008) The mother of all parasitesFuture Microbiology 3 391ndash395Page F C (1987) The classification of ldquonakedrdquo amoebae (PhylumRhizopoda) Archiv fuumlr Protistenkunde 133 199ndash217Parfrey LW Grant J Tekle Y I Lasek-Nesselquist EMorrison H G Sogin M L Patterson D J and Katz L A (2010)Broadly sampled multigene analyses yield a well-resolved eukaryotic tree oflife Systematic Biology 59 518ndash533Philippe H and Germot A (2000) Phylogeny of eukaryotes based onribosomal RNA long-branch attraction and models of sequence evolutionMolecular Biology and Evolution 17 830ndash834Philippe H Lopez P Brinkmann H Budin K Germot ALaurent JMoreira DMullerM and LeGuyader H (2000) Early-branching or fast-evolving eukaryotes An answer based on slowly evolvingpositions Proceedings of the Royal Society of London B Biological Sciences267 1213ndash1221Philippe H and Telford M J (2006) Large-scale sequencing and thenew animal phylogeny Trends in Ecology and Evolution 21 614ndash620Pusnik M Charriere F Maser P Waller R F Dagley M JLithgow T and Schneider A (2009) The single mitochondrial porin ofTrypanosoma brucei is the main metabolite transporter in the outermitochondrial membrane Molecular Biology and Evolution 26 671ndash680Reeb V C Peglar M T Yoon H S Bai J R Wu M Shiu PGrafenberg J L Reyes-Prieto A Rummele S E Gross J andBhattacharya D (2009) Interrelationships of chromalveolates within abroadly sampled tree of photosynthetic protistsMolecular Phylogenetics andEvolution 53 202ndash211Reyes-Prieto A Moustafa A and Bhattacharya D (2008) Multiplegenes of apparent algal origin suggest ciliates may once have beenphotosynthetic Current Biology 18 956ndash962Richards T A and Cavalier-Smith T (2005) Myosin domainevolution and the primary divergence of eukaryotesNature 436 1113ndash1118Rodriguez-Ezpeleta N Brinkmann H Burey S C Roure BBurger G Loffelhardt W Bohnert H J Philippe H andLang B F (2005) Monophyly of primary photosynthetic eukaryotesgreen plants red algae and glaucophytes Current Biology 15 1325ndash1330Roger A J Clark C G and Doolittle W F (1996) A possiblemitochondrial gene in the early-branching amitochondriate protist

Trichomonas vaginalis Proceedings of the National Academy of ScienceUSA 93 14618ndash14622Roger A J Svard S G Tovar J Clark C G Smith MWGillin F D and Sogin M L (1998) A mitochondrial-like chaperonin60 gene in Giardia lamblia Evidence that diplomonads once harbored anendosymbiont related to the progenitor of mitochondria Proceedings of theNational Academy of Sciences USA 95 229ndash234Rogozin I B Basu M K Csuros M and Koonin E V (2009)Analysis of rare genomic changes does not support the unikont-bikontphylogeny and suggests cyanobacterial symbiosis as the point of primaryradiation of eukaryotes Genome Biology and Evolution 1 99ndash113Sanchez Puerta M V and Delwiche C F (2008) A hypothesisfor plastid evolution in chromalveolates Journal of Phycology 44 1097ndash1107Saunders G and Hommersand Mh (2004) Assessing red algalsupraordinal diversity and taxonomy in the context of contemporarysystematic data American Journal of Botany 91 1494ndash1507Shadwick L L Spiegel FW Shadwick J D Brown MW andSilberman J D (2009) Eumycetozoa=Amoebozoa SSUrDNA phylo-geny of protosteloid slime molds and its significance for the amoebozoansupergroup PLoS One 4 e6754Shalchian-Tabrizi K Minge M A Espelund M Orr RRuden T Jakobsen K S and Cavalier-Smith T (2008)Multigene phylogeny of choanozoa and the origin of animals PLoS One3 e2098Simon N Cras A L Foulon E and Lemee R (2009) Diversityand evolution of marine phytoplankton Comptes Rendus Biologies 332159ndash170Simpson A G (2003) Cytoskeletal organization phylogenetic affinitiesand systematics in the contentious taxon Excavata (Eukaryota) InternationalJournal of Systematic and Evolutionary Microbiology 53 1759ndash1777Slamovits C H and Keeling P J (2008) Plastid-derived genes in thenonphotosynthetic alveolate Oxyrrhis marina Molecular Biology andEvolution 25 1297ndash1306Smith R and Patterson D (1986) Analyses of heliozoan interrelation-ships an example of the potentials and limitations of ultrastructuralapproaches to the study of protistan phylogeny Proceedings of the RoyalSociety of London B Biological Sciences 227 325ndash366Snow RW Guerra C A Noor AM Myint H Y and Hay S I(2005) The global distribution of clinical episodes of Plasmodiumfalciparum malaria Nature 434 214ndash217Sogin M L (1991) Early evolution and the origin of eukaryotes CurrentOpinions in Genetics and Development 1 457ndash463Sogin M L and Silberman J D (1998) Evolution of the protists andprotistan parasites from the perspective of molecular systematicsInternational Journal for Parasitology 28 11ndash20Stechmann A and Cavalier-Smith T (2002) Rooting the eukaryotetree by using a derived gene fusion Science 297 89ndash91Steuernagel B Taudien S GundlachH SeidelM Ariyadasa RSchulte D Petzold A Felder M Graner A Scholz UMayer K F Platzer M and Stein N (2009) De novo 454 sequencingof barcoded BAC pools for comprehensive gene survey and genome analysisin the complex genome of barley BMC Genomics 10 547Takishita K Yamaguchi H Maruyama T and Inagaki Y (2009)A hypothesis for the evolution of nuclear-encoded plastid-targetedglyceraldehyde-3-phosphate dehydrogenase genes in ldquochromalveolaterdquomembers PLoS One 4 e4737Taylor F J (1999) Ultrastructure as a control for protistan molecularphylogeny American Naturalist 154 S125ndashS136Tovar J Leon-Avila G Sanchez L B Sutak R Tachezy JVan Der Giezen M Hernandez M Muller M and Lucocq JM(2003) Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation Nature 426 172ndash176Van Der Giezen M (2009) Hydrogenosomes and mitosomes conserva-tion and evolution of functions Journal of Eukaryotic Microbiology 56 221ndash231Walker G Dacks J B and Embley TM (2006) Ultrastructuraldescription of Breviata anathema n gen n sp the organism previouslystudied as ldquoMastigamoeba invertensrdquo Journal of Eukaryotic Microbiology 5365ndash78Whittaker R H (1969) New concepts of the kingdoms of organismsScience 163 150ndash159Worden A Z Lee J H Mock T Rouze P Simmons M PAerts A L Allen A E Cuvelier M L Derelle E Everett M VFoulon E Grimwood J Gundlach H Henrissat B Napoli CMcdonald SM Parker M S Rombauts S Salamov A VonDassow P Badger J H Coutinho PM Demir E Dubchak IGentemann C Eikrem W Gready J E John U Lanier W



Lindquist E A Lucas S Mayer K F Moreau H Not FOtillar R Panaud O Pangilinan J Paulsen I Piegu BPoliakov A Robbens S Schmutz J Toulza E Wyss TZelensky A Zhou K Armbrust E V Bhattacharya DGoodenough UW Van De Peer Y and Grigoriev I V (2009)Green evolution and dynamic adaptations revealed by genomes of themarine picoeukaryotes Micromonas Science 324 268ndash272Yadav V P Mandal P K Rao D N and Bhattacharya S (2009)Characterization of the restriction enzyme-like endonuclease encoded by the

Entamoeba histolytica non-long terminal repeat retrotransposon EhLINE1FEBS Journal 276 7070ndash7082YoonH S Hackett J D Ciniglia C Pinto G and Bhattacharya D(2004) A molecular timeline for the origin of photosynthetic eukaryotesMolecular Biology and Evolution 21 809ndash818Yubuki N and Leander B S (2008) Ultrastructure and molecularphylogeny of Stephanopogon minuta an enigmatic microeukaryote frommarine interstitial environments European Journal of Protistology 44241ndash253



organisms A result in Plasmodium might be appli-cable toEimeria a finding inTrypanosoma bruceimaybe of relevance in Leishmania aetheopicaThis comparative approach means delving into

fields initially appearing tangential to parasitologyOur current understanding of eukaryotic systematicsis based both onmolecular andmicroscopic evidenceConsequently molecular phylogenetics and genomicsare playing an increasingly important roleEvolutionary cell biology and ancient eukaryoticevolution are two of the high-profile fields dependenton eukaryotic systemics They in turn are crucial forthe interpretation of parasitological and cell biologi-cal data in this comparative frameworkIn this review we will discuss the latest literature

on higher-level protist systematics and provide anupdated scheme of the major divisions of eukaryoticdiversity Each of these divisions will be describedhighlighting some of the parasitic organisms and thecurrent and upcoming genome projects Finally wewill explore two of the most pressing controversies ineukaryotic systematics and discuss the anticipatedrole that next generation sequencingmight play in theevolutionary study of parasites and eukaryotes ingeneral

SHIFTING VIEWS ON EUKARYOTIC SYSTEMATICS

FROM lsquoPRIMITIVE rsquo PROTOZOA TO DIVERSE

SUPERGROUPS

The tree of eukaryotes perhaps most familiar to manyparasitologists is that of a collection of crowneukaryotes (animals plants fungi and many algae)with a ladder-like sequential divergence of prominentparasites such as Giardia Trichomonas Encephalito-zoon and Trypanosoma from the base This view ofeukaryotic relationships and the place of parasites init was based on analyses of SSU rDNA genes andsingle protein coding genes performed during theearly to mid-1990s (Sogin 1991 Sogin and Silber-man 1998) Such a phylogeny very much supportedthe Archezoa hypothesis (Cavalier-Smith 19831987) a prevailing paradigm at the time (and stillone sometimes held today) that these ldquoamitochondri-aterdquo protists represent basal eukaryotic lineages thatdiverged before the acquisition of the mitochondrialendosymbiont and other key eukaryotic innovations(eg introns Golgi bodies peroxisomes) If truesuch parasites would be considered primitive andcould be used as possible routes to investigateancestral states of cellular systemsDespite its elegance and logic the Archezoa

hypothesis and the crownbase view of eukaryoticrelationships have been rejected based on several linesof evidence First of all mitochondrially-derivedorganelles (ie hydrogenosomes andmitosomes) havebeen found in nearly all of the proposed ldquoamito-chondriaterdquo organisms (van der Giezen 2009)Initially this was demonstrated by the identifying

of gene sequences of mitochondrial origin (HSP60HSP70 IscU) and later the localizing of these geneproducts (by immuno-microscopy) to double mem-brane-bound organelles found in these taxa (Clarkand Roger 1995 Bui et al 1996 Roger et al 19961998 Tovar et al 2003) Secondly it has been shownthat systematic phylogenetic error such as lsquolongbranch attractionrsquo has had a major effect on thepositioning of these organisms in the tree (Philippeand Germot 2000 Dacks et al 2002) The lessconserved gene sequences were clustered togetherregardless of whether the divergence was due to rapidevolution (as in many parasitic taxa) or a protractedperiod of time in which to accumulate independentmutations (as in the prokaryotic sequences used asoutgroups for the eukaryotic analyses) This artifactcaused otherwise highly divergent protists to bemistaken for basal eukaryotes When accounted forby algorithms andmodels that take differentmodes ofsequence evolution into account (Holder and Lewis2003) the lsquoprimitiversquo parasites and other proposedbasal eukaryotes were either clearly linked withrelatives elsewhere in the tree (as in the case ofMicrosporidia and fungi (Keeling and Fast 2002)) orwere unresolvedThe period following the demise of the Archezoa

hypothesis was one of taxonomic agnosticism andcaution with respect to interpretation of molecularsequence data and the evolution of eukaryotes Suchdata were still of immense value but a growingnumber of researchers abandoned the search for asingle gene that would resolve relationships greatand small in favour of a strategy whereby differentgenes were used to test hypotheses about particulartaxonomic relationships (eg EF2 demonstrating themonophyly of red and green algae (Moreira et al2000) or actin phylogenies uniting the cercozoans andforaminiferans (Keeling 2001)) These resolvedrelationships would then contribute to a consensusview of the eukaryotic tree as a whole It was also atime for a renewed appreciation of ultrastructuraldata and the realization of the need for this to beinterpreted in the light of molecular results (Taylor1999)These ideas crystallized in a seminal paper by

a consortium of taxonomic experts in the diverseprotist organisms An interim taxonomy of eukary-otes was thus provided by Adl et al (2005) based onmorphological ultrastructural and molecular dataand split the tree into six lsquosupergroupsrsquo Opistho-konta Amoebozoa Excavata ArchaeplastidaRhizaria and Chromalveolata By and large thesedivisions have held up and form the basis for thesupergroups that we will describe belowNonetheless work carried out in the 5 years since

the Adl et al paper necessitates changes in thesystematic viewpoint and eventually in the taxo-nomy In some cases the first gene sequences for keytaxa have been obtained shifting their affiliations











































































lsquoAmoebaersquo





















Metamonads














Discoba










Chlorophytes















Streptophytes
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES
























































































lsquoAmoebaersquo





















Metamonads














Discoba










Chlorophytes















Streptophytes
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES












































































lsquoAmoebaersquo





















Metamonads














Discoba










Chlorophytes















Streptophytes
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES
















































lsquoAmoebaersquo





















Metamonads














Discoba










Chlorophytes















Streptophytes
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES


































lsquoAmoebaersquo





















Metamonads














Discoba










Chlorophytes















Streptophytes
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES




























lsquoAmoebaersquo





















Metamonads














Discoba










Chlorophytes















Streptophytes
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES































Metamonads














Discoba










Chlorophytes















Streptophytes
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES




























Metamonads














Discoba










Chlorophytes















Streptophytes
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES























Discoba










Chlorophytes















Streptophytes
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES


















Discoba










Chlorophytes















Streptophytes
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES



























Streptophytes
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES
























Streptophytes
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES



















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES
















Rhodophytes


































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES

































Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES





















Filosa























Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES






















Endomyxa






























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES



























































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES


















































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES































CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES




















CONCLUSIONS








ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES



















ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES








































Documents

Eukaryotic systematics: a user s guide for cell biologists ... · Eukaryotic systematics: a user’s guide for cell biologists and parasitologists GISELLE WALKER1*, RICHARD G. DORRELL2†,