sign in sign up
<<
Home , Anaeromonadea, Anisonema, Blastocrithidia, Bodonida, Bodo (excavate), Calkinsia

Accepted Manuscript

Title: Higher Classification and Phylogeny of

Author: Thomas Cavalier-Smith

PII: S0932-4739(16)30083-9 DOI: http://dx.doi.org/doi:10.1016/j.ejop.2016.09.003 Reference: EJOP 25453

To appear in:

Received date: 2-3-2016 Revised date: 5-9-2016 Accepted date: 8-9-2016

Please cite this article as: Cavalier-Smith, Thomas, Higher Classification and Phylogeny of Euglenozoa.European Journal of Protistology http://dx.doi.org/10.1016/j.ejop.2016.09.003

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final . Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Higher Classification and Phylogeny of Euglenozoa

Thomas Cavalier-Smith

Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK

Corresponding author: e-mail [email protected] (T. Cavalier-Smith).

Abstract Discoveries of numerous new taxa and advances in ultrastructure and sequence phylogeny (including here the first site-heterogeneous 18S rDNA trees) require major improvements to euglenozoan higher-level . I therefore divide Euglenozoa into three subphyla of substantially different body plans: Euglenoida with pellicular strips; anaerobic Postgaardia ( Postgaardea) dependent on surface and with uniquely modified feeding apparatuses; and new Glycomonada characterised by glycosomes (Kinetoplastea, Diplonemea). Euglenoida comprise two new infraphyla: Entosiphona with three feeding rods and Dipilida ancestrally with two. Dipilida comprise superclass Rigimonada with longitudinal rigid strips [i.e. new classes Stavomonadea (Petalomonadida, Decastavida and new Heterostavida) and Ploeotarea (Ploeotiida) with contrasting oral ] and derived superclass Spirocuta with more numerous spirally arranged, often slideable, strips (clade Peranemea/Euglenophyceae) and a different, highly conserved pattern at strip joints. Peranemea comprise four orders: Peranemida (anterior gliding, protrusible rods), and three new, Anisonemida (posterior gliders), Natomonadida (swimmers including phagotrophic new suborder Metanemina and osmotrophic suborder Rhabdomonadina), and Acroglissida (anterior gliders with cytoproct). I establish orders Entosiphonida, Rapazida, Bihospitida; and seven new euglenoid families (Entosiphonidae, peranemean Neometanemidae, Rapazidae, two stavomonad, two ploeotiid) and three new postgaardid, and three kinetoplastid families (Neobodonidae, Rhynchomonadidae, Parabodonidae), plus new diplonemid Hemistasiidae for Hemistasia. Keywords: Diplonemea, Euglenoida, Glycomonada, Kinetoplastea, Spirocuta, Postgaardia

Introduction When establishing Euglenozoa to embrace euglenoids and kinetoplastids I argued that they differ in many respects from all other , and seriously considered putting them in a separate (Cavalier-Smith 1981) as suggested earlier (Cavalier-Smith 1978). They were ultrastructurally unique and the deepest branch on the first prokaryote-rooted protein sequence trees (mitochondrial cytochrome c: Schwartz and Dayhoff 1978). Euglenozoa are ancestrally aerobic, non-pseudopodial zooflagellates with a microtubule- rich pellicle, a unique complex feeding apparatus (FA), tubular extrusomes, parallel centrioles attached within a deep ciliary pocket by three distinctive microtubular roots to the pellicle and FA, and cilia ancestrally with unique dissimilar latticed paraxonemal rods (Cavalier-Smith 1981; Simpson 1997). More recent work amplifies their distinctiveness and confirms their evolutionary unity (Cavalier-Smith 2010a, 2013a). Of the six classes previously recognised, only Euglenophyceae secondarily acquired a green algal by (Cavalier-Smith 2013b; Gibbs 1981) and became that are well classified (Bicudo and Menezes 2016; ; Kim et al. 2010; Marin et al. 2003) except for there being no family or order for the highly distinctive phagotroph Rapaza (Yamaguchi et al. 2012). All other Euglenozoa are heterotrophic protozoa, phagotrophic or more rarely osmotrophic, symbiotrophic or parasitic, whose classification is currently confused and very incomplete. Mignot (1964) first noted paraxonemal rod similarities between a euglenoid and kinetoplastid, but they were first suggested as related because of similar (Leedale 1970), and recognized as a clade by Taylor (1976) and Cavalier-Smith (1978, both separated from other as „kingdom Euglenoida‟) for these and other reasons. They have a unique cytochrome c with haem attached via one cysteine (Pettigrew et al. 1975), not two as in all other organisms, which also requires different biosynthetic machinery from the radically contrasting machineries used by bacteria and excavates on the one hand and higher eukarotes on the other; a novel single-protein non-bacterial machinery evolved in the last common protozoan ancestor of , fungi, , and chromists (Allen 2011; Cavalier-Smith 2010a). Euglenozoa have dozens of molecular properties that are unique among eukaryotes. Many of these are clearly secondary acquisitions that would not have been present in the ancestral eukaryote, but about a dozen have been interpreted as primitive bacteria-related characters that collectively suggest that the root of the eukaryote evolutionary tree may lie between Euglenozoa and all other eukaryotes (Cavalier-Smith 2010a,b). Some multiprotein trees are consistent with that root position (Lasek- Nesselquist and Gogarten 2013) though others are not, suggesting instead that it may be between discicristates (Euglenozoa plus ) and other eukaryotes (Raymann et al.

2015) or that discicristates plus (Eozoa sensu Cavalier-Smith et al. 2015a) may be a clade (e.g. Derelle et al. 2015) not the ancestral eukaryote group as Cavalier-Smith (2010a,b) argued. The uniqueness of Euglenozoa is further emphasised by the remarkable discovery that euglenoid and kinetoplastid mitochondrial respiratory chains share 34 proteins absent from all other eukaryotes and bacteria (Perez et al. 2014). This respiratory chain uniqueness and their unique cytochrome c biogenesis (Allen 2011) would both have arisen immediately after the origin of mitochondria if the divergence between Euglenozoa and all other eukaryotes was the primary one in eukaryote evolution as Cavalier-Smith (2010a,b, 2013a, 2014b) argued is most likely, or somewhat later if the root were elsewhere. Euglenozoa now comprise four ultrastructurally very distinct types of phylogenetically related protozoan : euglenoids, postgaardids, diplonemids, and kinetoplastids (Cavalier-Smith 1998; Simpson 1997). Higher classification of Euglenozoa and euglenoids was last seriously revised two decades ago when three euglenoid classes were established within subphylum Euglenoida and diplonemids and kinetoplastids treated as separate single-class subphyla (Cavalier-Smith 1993). That revision was largely based on the structure of the euglenoid and diplonemid FA (Triemer and Farmer 1991a,b) and its phylogenetic implications (Cavalier-Smith 1995), but did not adequately evaluate ultrastructural aspects of euglenoid pellicle morphogenesis (Belhadri and Brugerolle 1992; Mignot et al. 1987; Triemer and Fritz 1988). It also preceded discovery of (Fenchel et al. 1995) with novel ultrastructure placing it in Euglenozoa (Simpson et al. 1996/7), where it was formally accomodated within new class Postgaardea grouped with kinetoplastids as subphylum Saccostoma (Cavalier-Smith 1998). Currently, Euglenozoa are classified in subkingdom Eozoa of kingdom Protozoa and subdivided into six classes (three in subphylum Euglenoida: Peranemea, Euglenophyceae, and one unnamed) and 12 orders, subphylum Saccostoma being abandoned because Postgaardea proved to be much more distinctive than was originally recognised (Ruggiero et al. 2015). As recently revised (Cavalier-Smith et al. 2015a), Eozoa include only three phyla, phenotypically radically different and genetically deeply divergent but internally relatively uniform: (1) Euglenozoa with ciliary paraxonemal rods; (2) the biciliate grooved Eolouka (i.e. jakobids with a vaned posterior cilium and the most primitive mitochondrial genomes, plus Tsukubamonas without ciliary vanes); and (3) the quadriciliate Percolozoa without paraxonemal rods or vanes but with unstacked Golgi membranes (unlike Euglenozoa and most eukaryotes other than higher fungi and ). Percolozoa share discoid mitochondrial cristae with Euglenozoa so these discicristates were once made an infrakingdom (Cavalier-Smith 1998). The presence of a few respiratory chain proteins shared by Euglenozoa and Percolozoa (Perez et al.

2014), some of which might conceivably be involved in causing their discoid cristal form, but which are absent from higher eukaryotes and bacteria, makes it possible that (or Eozoa if also present in Eolouka, not currently known) are a clade, implying that the eukaryote root is not between Euglenozoa and Percolozoa unless these are ancestral proteins lost by other eukaryotes. Previously Eolouka were lumped with that has a cytoskeletally related feeding groove (but a positionally non-homologous posterior ciliary vane) as phylum Loukozoa (Cavalier-Smith 1999), which was later grouped with Percolozoa and the anaerobic, ancestrally quadriciliate Metamonada as infrakingdom (Cavalier-Smith 2010a; Ruggiero et al. (2015). However multigene trees repeatedly showed that Malawimonas and Metamonada are more closely related to the podiate clade that includes , , and Sulcozoa (Cavalier-Smith 2013a) than to Eolouka or Euglenozoa, and that excavates are a paraphyletic organizational grade (Brown et al. 2013; Burki et al. 2016; Cavalier-Smith et al. 2014, 2015a,b, 2016). Therefore Malawimonas and Metamonada were formally transferred from subkingdom Eozoa to protozoan subkingdom Neozoa by Cavalier- Smith et al. (2015a), Excavata (originally made an infrakingdom by Cavalier-Smith 2002) being abandoned as a taxon, a substantial rearrangement of kingdom Protozoa that better fits eukaryote deep phylogeny and the distribution of the two types of ciliary vanes on the eukaryote tree. Of particular importance for overall euglenozoan taxonomy is the demonstration that , originally considered a euglenoid (Lackey 1960), is ultrastructurally more similar to Postgaardi (Yubuki et al. 2009, 2013), thus confirming the placement of Calkinsia in class Postgaardea by Cavalier-Smith (2003 a,b), which was not accepted by Adl et al. (2005) or by Yabuki et al. (2009) who proposed a new, in my view unnecessary, clade name Symbiontida for Calkinsia plus unspecified relatives, then excluding Postgaardi. The recently discovered postgaardean Bihospites (Breglia et al. 2010) revealed novel ultrastructural features confirming the distinctiveness of Postgaardea/Symbiontida. Though Adl et al. (2012) followed Cavalier- Smith (1998, 2003a,b) in treating them as a group distinct from Euglenoida, Lax and Simpson (2013), Lee and Simpson (2014a,b), and Chan et al. (2015) reverted to including all Postgaardea in euglenoids as a discrete subgroup. For euglenoids sensu stricto (i.e. excluding Postgaardea), a recent system (Adl et al. 2005, 2012) reverted to Klebs‟ (1892) oversimplified three- group system, ignoring numerous advances over the past century; there is a pressing need to use them to make a far better classification, especially for the phagotrophs. Similarly important was the demonstration that kinetoplastid ultrastructural diversity is much greater than is even now generally appreciated (Frolov and Karpov 1995; Frolov et al. 2001), and certainly at the time of the previous major revision (Cavalier-Smith 1993) and when kinetoplastids were first compared in detail with euglenoids (Brugerolle 1985; Kivic and Walne 1984). Moreira et al. (2004) established an improved phylogenetic classification of kinetoplastids, which can readily accomodate the additional genera discovered since (e.g. Flegontov et al. 2013; Hirose et al. 2012; Stoeck et al. 2005). Diplonemid evolution and diplonemid/kinetoplastid relationships have been recently greatly clarified by the demonstration that Hemistasia previously considered a kinetoplastid (Adl et al. 2012; Elbrächter et al. 1996) is actually a diplonemid (Yabuki and Tame 2015). Many other advances over the past two decades, improved and taxonomically more comprehensive sequence trees (including those presented in this paper), and critical reevaluation of euglenozoan comparative anatomy (which I shall publish in more detail elsewhere), make a radically improved euglenozoan taxonomy now essential especially for the relatively neglected phagotrophic euglenoids and Postgaardea and at higher ranks. The purpose of this paper is to present such a revised classification (Table 1) comprehensive to the family level (placing almost all genera); to explain the reasons for the most important innovations and simplications; to provide enough historical background to put them into perspective; and to provide the first site-heterogeneous 18S rDNA tree for Euglenozoa (Fig. 1) to enable comparison of sequence phylogeny with the new classification. At the highest taxonomic level the major innovation is the new subphylum Glycomonada to embrace kinetoplastids and diplonemids, which 187-gene trees show are undoubtedly sister classes (Cavalier-Smith et al. 2014, 2015a,b, 2016b). The name emphasises that both classes have glycosomes that contain the glycolytic enzymes (Makiuchi et al. 2011; Gualdrón-López et al. 2012), in contrast to the homologous more typical peroxisomes of all other eukaryotes whose glycolysis takes place in the cytosol. Euglenoida remain a subphylum as in Cavalier-Smith (1993). Postgaardea are placed in the new subphylum Postgaardia, as they are radically different ultrastructurally from both euglenoids and glycomonads and are a third deep-branching euglenozoan clade that may be sister to Euglenoida but do not branch within them or Glycomonada on the evolutionary most realistic sequence trees presented in the next three sections, contrary to some poorly resolved earlier trees. Lower level innovations are predominantly amongst phagotrophic euglenoids as numerous ultrastructurally and phylogenetically distinctive new genera have been recently discovered or better characterised (Breglia et al. 2013; Cavalier-Smith et al. 2016a; Chan et al. 2013, 2015; Lax and Simpson 2013; Lee and Simpson, 2014a,b; Yamaguchi et al. 2012). This classification is the first to use families comprehensively since Euglenozoa was established; 14 of the 31 recognised here are new, as are seven of the 18 orders and three out of eight classes. Families were entirely omitted by Leedale (1967, 2002) for euglenoids. Indeed, all works on heterotrophic euglenoids known to me since Vasileva (1987) ignored them; that deficiency reflects the difficulty of delimiting euglenoid families noted by Pringsheim (1948) who rightly stressed that the three dating from Klebs (1892) - Euglenidae (), Astasiidae (osmotrophs), and Peranemidae (phagotrophs) - 'do not, however, seem to coincide with true taxonomic groups'. Fortunately, comparative ultrastructure and better taxon-rich sequence trees (next ) now allow definitions of euglenoid families with good prospects of future stability, as already well done for non-phagotrophic phototrophs (Marin et al. 2003; Kim et al. 2010b) and essayed here for the rest. I discuss classification of each subphylum in sequence, starting with euglenoids, after first explaining my rather comprehensive site-heterogeneous sequence trees.

Euglenozoan site-heterogenous 18S rDNA phylogeny: Rationale and methods Using macgde v. 2.4 (http://macgde.bio.cmich.edu/) I manually aligned 18S rDNAs of 217 Euglenozoa plus 481 outgroup taxa representing all major eukaryote groups and selected by eye 1577 or 1541 reasonably well aligned nucleotide positions for preliminary phylogenetic analysis (>50% more than in some euglenozoan studies), depending on whether the highly divergent Percolozoa were excluded or included. 18S rDNA of Percolozoa, sometimes included as outgroups for euglenozoan phylogeny (Lax and Simpson 2013; von der Heyden et al. 2004), evolves much faster than in most eukaryotes making its alignment especially difficult and potentially introducing long-branch artefacts (Cavalier-Smith 2015). Ribosomal DNA trees for Euglenozoa have typically been basally poorly resolved and contradictory for deep relationships (Chan et al. 2013; Lax and Simpson 2013; von der Heyden et al. 2004; Yubuki et al. 2009). To increase resolution I did four things: (1) used not only maximum likelihood (ML) as in most other studies, but also the CAT-GTR model of PhyloBayes, which is evolutionarily more realistic in allowing different patterns of nucleotide substitution across sites in the molecule (a site-heterogeneous model, not site-homogeneous as in ML (Lartillot and Philippe 2004); ML can be misleading if unrealistic models are used) and should give more accurate trees (never previously done for Euglenozoa), as it appears to have done for other difficult to resolve protist groups with rDNA long-branches such as gregarines and Percolozoa (Cavalier-Smith 2014a, 2015); (2) included many more sequences than any previous study, which should increase accuracy of ancestral state reconstruction; (3) included a much higher proportion of the 18S rDNA molecule than before to provide more phylogenetically useful characters (up to 1577 nucleotides, over 50% more than in some studies) made possible by including so many taxa, which enables accurate alignment in regions previously excluded because alignment is harder when taxa are sparser; and (4) included many more outgroup taxa than before, which should enable the position of the euglenozoan tree‟s root to be more accurately calculated, reducing potential errors from its misplacement. Preliminary analysis investigated effects of outgroup choice on euglenozoan phylogeny for the first time by analyzing by both methods three different taxon and gene segment samples. Since von der Heyden et al (2004) it has been customary to use only excavates as outgroups in euglenozoan rDNA trees; Lax and Simpson (2013) used Jakobea, Tsukubamonas and Percolozoa, whereas others used even more restricted outgroups: just two Jakobina (Chan et al. 2013) or two Andalucina (Yubuki et al. 2009); most used no outgroups so were arbitrarily rooted (e.g. Breglia et al. 2013; Lee and Simpson 2014a,b; Yamaguchi et al. 2012). Restricted outgroups (especially just two of one suborder) risk biasing the position of the euglenozoan root by being unrepresentative; a problem with including Percolozoa is that they have systematically the longest branches of any whole eukaryotic phylum (Cavalier-Smith 2014a, 2015) and risk biasing the root position by convergent evolution with long ingroup branches. A broadly representative outgroup without excessively long branches is probably best; as many non-excavates have shorter branches and are evolutionary diverse, excluding them in previous studies was unwise. I initially analysed a 698-taxon sample including 382 short-branch representatives of all non-excavate outgroups, 43 short-branch excavates, and a thorough sampling of Percolozoa, but with only nucleotides accurately alignable with them included (1541 nucleotides) to allow comparison with previously published trees using only outgroups; and a 609 taxon sample with 382 short-branch representatives of all neokaryote outgroups other than Percolozoa. To allow faster convergence of PhyloBayes trees in later analyses I pruned outgroup diversity of close relatives without sacrificing phylogenetic breadth. The two PhyloBayes chains in the resulting 240 taxon sample (supplementary Fig. S1) converged well onto the same topology (maxdiff 0.16). Convergence was less good for the 609 and 698 taxa samples; nearly all their branch patterns were the same, but there were a few differences, especially near the base of Euglenozoa. There were significantly more differences with ML. To reduce discrepancies I added ~35 more Euglenozoa to the alignment, removed the longest branch Percolozoa (mainly Percolatea) and further improved parts of the alignment, mainly to certain partially misaligned Entosiphon segments, but in small ways also to some relatives of added Euglenozoa. To lessen computing time and ensure convergence in this improved alignment I restricted sampling of non-eozoans to a representative subset, giving 323 taxa altogether; as Entosiphon has the longest branch in the tree, I also analysed 318 taxa excluding Entosiphon to see if its presence distorted topology or reduced bootstrap support for bipartitions of interest. I also ran trees excluding all Percolozoa to check whether their divergent sequences change the apparent position of the euglenozoan root (282 taxa without and 287 taxa with Entosiphon: Fig. 1). For accurate topology among closer relatives one should include as many nucleotide positions as possible, whereas for accuracy for deep branches it may be better to exclude some of the fastest evolving sites (unfortunately there is probably no overall optimal number and no objective way of deciding a best compromise). For our first Euglenozoa-wide trees (von der Heyden et al. 2004) we included only 1233 positions, but much improved taxon sampling now allows me to align 1577 nucleotides with reasonable confidence. To check whether excluding the fastest evolving (but generally reasonably aligned) sites gives different results, I also ran trees including only 1425 nucleotides. Phylogenetic analysis was by RAxMLHPC-PTHREADS-SSE3 v. 7.3.0 (Stamatakis 2006) using the GTRGAMMA model with four rate categories and 400 or 1000 fast bootstraps (4 processors) and by the site-heterogeneous CAT-GTR-GAMMA (4 rates) model of PhyloBayes v. 3.3 (Lartillot and Philippe 2004) with two chains for thousands of generations after log likelihood values plateaued, early pre-plateau trees being removed as burnin before summation of all other trees (for brevity called CAT only in the text). Using the final improved rDNA alignment I ran 16 trees with different taxon and sequence samples and algorithm in all the above combinations. All Bayesian trees converged with maxdiff <0.3, mostly 0.1 or less. I prepared trees for publication using FigTree v. 1.2.2 (http://tree.bio.ed.ac.uk/software/figtree/) and Eazydraw. I compared my 18S rDNA alignment to the 1374 nucleotide alignment of Chan et al. (2015); that is grossly misaligned in several tracts, especially for Serpenomonas (=Ploeotia costata) the subject of their paper. Moreover it included many nucleotide positions here excluded (from both my 1425 and 1577 nucleotide selections) because they cannot be accurately aligned or seem too divergent, but excluded several substantial segments that I included and which are not especially difficult to align, quite well conserved, and phylogenetically informative. These serious alignment errors and non-ideal choice of segments for analysis probably explain why Chan et al. (2015) did not even show Euglenoida as monophyletic, as it is with strong support on all but one of my trees (an ML tree including the extremely long-branch Entosiphon; Euglenoida was invariably a clade with PhyloBayes). I did not make similar comparisons with other papers using widely varying nucleotide numbers, down to 734 (Yubuki et al. 2009), as their alignments were not available; however this comparison cautions against assuming that alignments including fewer nucleotides are necessarily more accurate. As my alignment took much effort and care, and is taxonomically and in nucleotide positions much more comprehensive than any other to date (and I suspect more accurate than most), it is in the supplementary material for general use and criticism (together with the three masks used for selecting positions for analysis).

Euglenozoan molecular phylogeny Figure 1 is a site-heterogeneous tree based on 1577 nucleotide positions of 18S rDNA with 224 Euglenozoa plus outgroups restricted to short-branch excavate lineages as in most previously published euglenoid rDNA trees (also for ease of fitting onto one page). It excluded long-branch Entosiphon whose position is plotted on it from a separately run tree with identical alignment. On the latter tree tree Entosiphon appears as sister to Stavomonadea (Fig. 1), whereas by Hsp90 it is sister to all Dipilida (Cavalier-Smith et al. 2016a). Supplementary Fig. S1 is a tree run before Neometanema parovale (Lee and Simpson 2014) and Serpenomonas costata sequences of Chan et al. (2015) were published, and before I improved Entosiphon's alignment to my present satisfaction; CAT misleadingly put Entosiphon within Stavomonadea as in Lax and Simpson (2013) but as sister to Keelungia not petalomonads as on their tree (and by ML for the Fig. S1 alignment). This shows unsurprisingly that some misalignment can change the apparent position of Entosiphon, and also that the relatively small degree of Fig. S1 misalignment (much less than in Chan et al. (2015)) predominently of Entosiphon does not change euglenozoan topology generally, with the single exception of the rather close basal branching order of the three stavomonad orders (different in Figs 1 and S1 CAT though S1 ML had the same stavomonad topology as Fig. 1) and of metakinetoplastids (Fig. S1 with a slightly less refined alignment wrongly put as sister to rather than within bodonids as in Figs 1, S2 and previous multiprotein trees). Extremely close bush-like branching of several lineages is generally the most important factor (more so than long-branches alone) that makes parts of sequence trees extremely hard to resolve, even with trees based on hundreds of proteins (Cavalier-Smith et al. 2015b). Supplementary Fig. S2 using 1577 nucleotide positions from 323 taxa including Entosiphon and a much broader and more representative outgroup selection including Percolozoa exemplifies the 16 trees obtained after the best alignment (also used with fewer taxa for Fig. 1) was established. It shows the same branching order for Stavomonadea as Fig. 1, differing within euglenoids only in the positions of Entosiphon and Teloprocta, each moving by one node, and also in the deep branching within Metakinetoplastina (though like Fig. 1 correctly placing trypanosomatids within bodonids). Fig. S2 shows Entosiphon as sister to Dipilida (therefore the deepest branching euglenoid) in complete harmony with Hsp90 (Cavalier-Smith et al. 2016a) but much lower posterior probablity support. However, even with this superior alignment the position of Postgaardea (Table 2), Entosiphon and Teloprocta (Table 3) are sensitive to whether or not Percolozoa is present in the outgroup, to algorithm (site- heterogeneous or not), and to whether more or fewer faster evolving parts of the molecule are included. In all other respects euglenozoan tree topology except deep metakinetoplastid topology was insensitive to these technical variations with this improved alignment with almost all nodes strongly or maximally supported (Fig. S2). Thus site-heterogeneous 18S trees with massive taxon sampling including broadly sampled outgroups give a better resolved deep euglenozoan branching order than previous less well sampled site-homogeneous trees. On all 18S rDNA trees Diplonemea and Kinetoplastea group together as a clade (here called Glycomonada because of their shared glycosomes; Fig. 1), but support varies greatly with different taxon samples, outgroups, and number of included nucleotides (maximally with posterior probability (PP) 0.99 and ML bootstrap support (BS) 88%). A concurrently published Hsp90 tree, though much more sparsely sampled (Cavalier-Smith et al. 2016a, fig. 9), is also congruent for both methods: Diplonemea are sisters to kinetoplastids with maximal support by CAT and near maximal (99%) by ML. This agreement between Hsp90 and rDNA trees contrasts with many previous 18S rDNA studies noted above that incorrectly grouped Diplonemea with Euglenoida not with Kinetoplastea as Hsp90 showed. Within Kinetoplastea there is maximal support for the deep divergence of prokinetoplastids and metakinetoplastids (bodonids plus trypanosomatids) for both molecules, but basal branching within metakinetoplastids is slightly contradictory between them in two respects: Hsp90 puts as sister to trypanosomatids, like 192-gene trees (Cavalier-Smith et al. 2014), whereas rDNA groups Bodo and parabodonids with insignificant support (most CAT trees, e.g. Figs 1, S1) or groups parabodonids and trypanosomatids with insignificant support (e.g. Fig. S2 CAT and Fig. 1 and other ML trees); Hsp90 shows neobodonids as paraphyletic whereas rDNA either agrees (ML; CAT with 1425 positions) or shows them as a weakly supported clade (CAT with 1577 positions, e.g. Figs 1, S2). The internal phylogeny of euglenoids shown by my present rDNA trees was discussed in detail by Cavalier-Smith et al. (2016a). In brief, they strongly confirm holophyly of Spirocuta with numerous spiral strips and that they are evolutionarily derived from (ancestral or paraphyletic) Rigimonada with fewer longitudinal strips and ancestrally a 2-rod FA. Entosiphon, also with few longitudinal strips but a radically distinct 3-rod FA is clearly the most deeply divergent euglenoid on Hsp90 trees; though some rDNA trees support this others do not (Table 2). All contradict earlier inclusion of Entosiphon in Peranemida (Cavalier-Smith 1993b), as does Hsp90 strongly. In most cases, inclusion of Entosiphon or not did not alter tree topology, just support values for deepest euglenoid branches. However its exceptionally divergent rDNA prevents its conclusive placement by rDNA sequence trees, making its position unstable and highly sensitive to taxon and site sampling, so Hsp90 is probably more reliable. The only rDNA tree that correctly placed it is the CAT tree with the most complete sequence and broadest outgroup representation (1577 positions, 323 taxa: Fig. S2); this shows that (provided one takes exceptional care with the alignment in a very large database) using the largest practical number of sites can be more accurate than trimming alignments drastically to include only the most easily aligned parts that is too often done (and never positioned Entosiphon concordantly with Hsp90 trees). The position of Teloprocta is also unstable as previously found (Table 2). Organisms called Ploeotia in the past are so deeply divergent that they clearly belong in several genera, e.g. Serpenomonas costata does not group with Ploeotia cf. vitrea, showing that phagotrophic euglenoids are much more diverse than often supposed. As noted below, Serpenomonas pellicle is unique but previously greatly misinterpreted, so Serpenomonas is here placed in a separate order distinct from order Decamonadida recently established for Decamonas and Keelungia (Cavalier-Smith et al. 2016a), and grouped with it and Petalomonadida as new class Stavomonadea that excludes Ploeotiida sensu stricto (Table 1). Stavomonadea are a clade on rDNA trees where Entosiphon is excluded or if included does not (arguably artefactually) intrude amongst them. Petalomonadida are weakly sister to Decamonadida (Figs 1, S1 ML, S2). Though multigene trees are needed to establish the branching order of the three stavomonad orders with confidence, their collective unity and consistent strong divergence from Ploeotia cf. vitrea on Figs 1, S1, S2 and all my other trees make it clearer than before that petalomonad FAs are secondarily simplified by rod loss, not primitively simple as often assumed, and suggest that their pellicles are also secondarily simplified (Cavalier-Smith et al. 2016a).

Postgaardea are not euglenoids, but possibly their sisters Unlike some published ones, my 18S rDNA trees collectively argue strongly against the bacteria-covered postgaardids branching within Kinetoplastea or classical euglenoids. Contrary to previous conflicting studies, all except one rDNA tree firmly excluded Postgaardea (=Symbiontida) from euglenoids, which are a robust clade with support for exclusion of Postgaardea varying with sampling (maximally 0.99, 80%). However the position of postgaardeans is sensitive to outgroup choice and algorithm. Amongst 16 trees for the most refined alignments, CAT always showed Postgaardea as sister of euglenoids with typically insignificant support (PP 0.44-67; details Table 3); four ML trees also gave that topology (BS 40-70%); three contradictorily put them as sister to Glycomonada (25-32%) and one as sister of

Entosiphon only (30%). All preliminary trees also strongly excluded them from Euglenoida. The unique grouping with Entosiphon is clearly artefactual. A relationship with euglenoids is likely (and consistent with most previously published ML trees), but protein trees are needed to decide between the two alternatives and the third possibility that Postgaardea are sisters of all other Euglenozoa, which rDNA trees do not exclude. Critical pellicle comparisons lead me to conclude that the Bihospites pellicle does not have euglenoid-like S-shaped pellicular strips as Breglia et al. (2010) claimed. Postgaardid pellicle local ultrastructure is essentially the same as in diplonemids and gives no support to a relationship with euglenoids; Simpson et al. (1996/7) stressed that the even mt array of Postgaardi pellicles is „indistinguishable from the pattern seen in large kinetoplastids and diplonemids‟ and „do not follow the “ pattern”‟. Bihospites and Calkinsia pellicles have essentially identical mt arrangement and cross-bridging. In my view FA homologies of Postgaardea have also been misinterpreted, as briefly explained in a later section. Fig. 2 summarises the findings of rDNA and Hsp90 trees.

Euglenoid high-level taxonomy needs radical revision Bütschli (1884) first established euglenoids as an almost unified group under the name Euglenoidina (not Euglenida as used by Adl et al. 2012 who claim to select the oldest names). Lankester (1885) included six families - only five actually euglenoids and only one (Euglenina) photosynthetic - in order Euglenoidea Bütschli, still the most suitable spelling if euglenoids are treated as a class (e.g. Cavalier-Smith 2003a,b; Hausmann and Hülsmann 1996). Lankester also had a fourth phagotrophic euglenoid family, Anisonemina comprising and Entosiphon (narrower than Anisonemidae Saville Kent, 1880 that also had ), but did not consider them euglenoids, placing it instead in Bütschli's now defunct order Heteromastigoda together with a much more heterogeneous family Bodonina of heterotrophic flagellates including Bodo, Heteromita, and four non-euglenozoan genera. For a century classification was dominated by a simpler, less accurate into just three families (Klebs 1892) that lumped Lankester's four phagotrophic families into one: phototrophs (Euglenida, i.e not all euglenoids), osmotrophs (Astasiida) and phagotrophs (Peranemida), as exemplified by the first comprehensive treatise on euglenoids in the modern sense (Walton 1915), though some authors had two or three photosynthetic families (Hollande 1942; Smith 1933). Hollande (1942) split euglenoids primarily into three, but unlike Klebs grouped osmotrophs (2 families) with three families of phototrophs (=Aphagea of Cavalier-Smith 1993) and split phagotrophs into two: Péranémöidinées and Pétalomonadinées, the latter including Ploeotia, Entosiphon, and

Anisonema with petalomonads sensu stricto. His later superior system (Hollande 1952) like Lankester's separated Petalomonadidae and Anisonemidae (i.e. Anisonema, Entosiphon) and put Ploeotia (omitted by Lankester and Saville Kent) . In the twentieth century some phycologists initially ignored euglenoids (West 1904), following Klebs (1883) who considered them protozoan flagellate Euglenoidina not algae. But others embraced them as Euglenineae (Senn 1900; later a class: West and Fritsch 1932; Fritsch 1948) or Eugleninae (Lemmerman 1913; later a class, Shoenichen 1925) or division Euglenophyta with two classes (Pascher 1931). Eventually phycologists settled on spelling the class Euglenophyceae (Huber-Pestalozzi 1955; Smith 1933) and consistently using euglenoid in the vernacular (Leedale 1967; Triemer and Farmer 2007). Protozoologists in contrast long treated all flagellates as just one class, slavishly following Bütschli, so ranked euglenoids only as an order - spelt Euglenoidina (e.g. Walton 1915 with families Euglenidae, Astasiidae, Peranemidae) or Euglenida, e.g. Calkins (1926) also with three families, photosynthetic Euglenidae, phagotrophic Heteronemidae (contrary to Adl et al. 2005, 2012, Heteronemina Leedale, 1987 was not its first use as a suprageneric taxon), and Astasiidae with a mix of osmotrophs and phagotrophs including both and - a more heterogeneous mixture than Lankester's Astasiidae, which excluded Peranemidae and Petalomonadidae as separate families. A protozoological committee (Honigberg et al. 1964) divided order Euglenida into suborders Euglenina, Peranematina and Petalomonadina, clearly based on Hollande (1942 not 1952). Given so many past conflicting spellings, the most sensible protistological compromise is to harmonise vernacular and formal terms by uniformly using the oldest vernacular term euglenoid for the whole group and Euglenoida for their subphylum (Table 1 and Cavalier-Smith 1978, 1993), as in Ruggiero et al. (2015). Electron microscopy provided germs of a more fundamental subdivision in the six orders or suborders of Leedale (1967) still extant in some publications, e.g. Leedale (2002) where it remained deficient in having no families. Leedale (1967) retrogressed somewhat by lumping petalomonads and Anisonema as Sphenomonadina, a new name compositionally equivalent to Pétalomonadinées of Hollande (1942), and adopting Heteronematina in an only slightly narrower sense than Calkins (1926) extremely heterogeneous Heteronematidae (by segregating Sphenomonadina). Levine et al. (1980, with Leedale coauthor) accepted his six suborders. Cavalier-Smith (1993) abandoned Sphenomonadales/-ina as too heterogeneous, using the older name when establishing order Petalomonadida that like Petalomonadidae sensu Hollande (1952) excluded Ploeotia, Entosiphon, and Anisonema, and segregated a new order Ploeotiida from Peranemida; for the first time Euglenoida was ranked as a protozoan subphylum with three classes with contrasting feeding machinery: Aphagea, and the phagotrophic Petalomonadea and Peranemea. Adl et al. (2005) reverted to Klebs' (1892) oversimplified three-group system with no subordinate phagotrophic euglenoid suprageneric taxa, but changed two of his names. Instead of adopting his Peranemida for phagotrophs they used Heteronematina of Leedale (1967) by radically changing its meaning to including all Sphenomonadina (in this broadened sense it ought to have been called Heteronematidae Calkins, 1926 even though his group was narrower than theirs and different from Leedale's), and followed Busse and Preisfeld (2003) in restricting Aphagea to osmotrophs, i.e. a different meaning from the original (Cavalier-Smith 1993); both changes contravened their assertion that they „used the older name that describes each taxon unless its composition was substantially modified‟. Thus three generations of Society of Protozoologists/International Society of Protistology committee taxonomy (Adl et al. 2005, 2012; Honigberg et al. 1964; Levine et al. 1980) simply returned us to 1892 for euglenoids; the Adl et al. (2005) system was not „the new higher level classification‟ it proclaimed. Despite its first sentence wrongly saying Adl et al. (2005) „established name stability‟, Adl et al. (2012) without explanation changed circumscription of their name (Euglenea in 2005) by excluding Eutreptiales, and grouped Eutreptiales, Euglenea, and the photophagotroph Rapaza (Yamaguchi et al. 2012) with equal rank within Euglenophyceae. Unlike earlier systems (Cavalier-Smith 1993, 1998; Hollande 1952; Honigberg et al. 1964; Leedale 1967, 2002; Walton 1915) that of Adl et al. (2005, 2012) was not a balanced comprehensive classification with taxa employing Linnean categories like phylum, class and order; nor was it a strict cladifaction (Mayr and Bock 2002) as like traditional taxonomy it accepted some paraphyletic groups, though seeming to regard their elimination as intrinsically desirable (Adl et al. 2012 p. 430). Using only four ranks severely limited its usefulness - it unwisely gave the same rank to single genera like , , or (previously all placed sensibly by others in higher taxa) as to Euglenozoa, Fungi, all animals or all plants, a criticism also made by Ruggiero et al. (2015). Such idiosyncratic ranking gives as much weight to ignorance as to knowledge. Cladifications can be useful, but have different aims from and do not replace the function of traditional Linnean classification (Mayr and Bock 2002), i.e. to provide a user-friendly, hierarchical classification of all with taxa ranked according to phenotypic distinctiveness and arranged in an evolutionarily sound manner without polyphyletic groups, comprehensively at each rank (Cavalier-Smith 1998; Ruggiero et al. 2015). To this end Table 1 offers the product of a single mind integrating over a century of progress since pioneering Bütschli (1884) and Klebs (1883, 1892); I hope it provides a good basis for further improvement and debate. I need not defend establishing the ancestral superclass Rigimonada or retaining paraphyletic orders Eutreptiida and (all flagged in Table 1 as paraphyletic to prevent readers misinterpreting them as clades), as the standard arguments against ancestral (paraphyletic) taxa are unsound and refuted in detail elsewhere (Cavalier- Smith 2010b). However, those averse to such groups have the consolation that my changes make Petalomonas, Ploeotia, Heteronema, Ploeotiida, and Peranemida no longer paraphyletic on rDNA trees. To provide a hierarchical, ranked reference classification suitable for general use (Ruggiero et al. 2015) it is comprehensive for the standard categories of subphylum, class, order, and family, and introduces additional intermediate categories only for those subgroups where necessary to subdivide them appropriately. In places this necessarily includes intermediate categories that can be omitted by endusers whose purposes are better served by listing only classes (as in Fig. 2 centre column), only orders (Fig. 2 middle column) or only families within a particular group. Arteficially restricting the number of categories used for higher taxa like Entosiphona having only one by avoiding standard intermediate categories that some might imagine to be superfluous, would have deprived end users of that flexibility and also left some genera unassigned to families, orders or classes, which in itself could be a potential source of ambiguity or confusion that is best avoided. The notion expressed by one referee that one should never place a single genus alone in its own higher taxon is irrational and contrary to 300 years of taxonomic practice.

Five classes and seven new orders of euglenoids Though one might treat all euglenoids as one class (Cavalier-Smith 2003a,b; Hollande 1952; Smith 1933), general use of Euglenophyceae as a class for phototrophs only (Bicudo and Menezes 2016; Marin et al. 2003) accepts that dispensing with the classical distinction between green and colourless forms (Leedale 1967) was a mistake, and makes thorough reevaluation of phagotrophic euglenoid class demarcation overdue. Previously euglenoids were divided into three classes solely using feeding apparatus (FA) differences, before rDNA was sequenced for phagotrophs, so Ploeotiida and Entosiphonida were wrongly included in Peranemea (Cavalier- Smith 1993). The five euglenoid classes here, and grouping two as superclass Spirocuta, better express euglenoid megadiversity and phylogeny than the former three (Cavalier-Smith 1993). One, long accepted Euglenophyceae, is identical to one of Klebs (1892) three groups; the other four reflect much improved undertanding of phagotrophic euglenoids and their relationships in the past 20 years, including evidence that rhabdomonads are more closely related to some of them than to Euglenophyceae, and a fundamental reevaluation of pellicle and FA comparative anatomy (Cavalier-Smith unpublished), so are evolutionarily more realistic.

Adl et al. (2012) did not subclassify euglenoid phagotrophs, but a footnote accepts Petalomonadida as probably a clade, but suggests expanding Ploeotiida to include Entosiphon, Lentomonas, and Keelungia with Ploeotia. However, putting Entosiphon in Ploeotiida (von der Heyden et al. 2004) made it too heterogeneous in FA structure for an order and was little improvement on grouping it with Anisonema (Saville Kent 1880-1881; Lankester 1885) with which it was originally congeneric (Dujardin 1841), so I establish a separate order Entosiphonida. Even the remaining Ploeotia-like taxa are not ultrastructurally similar enough for one order, which would be radically broader than the original Ploeotiida. Such a group would be deeply paraphyletic and lack a clear unifying morphology. All recent sequence trees show Petalomonadida as a robust clade (Figs 1, S2 and for Hsp90 Cavalier-Smith et al. 2016a), confirm the polyphyly of Sphenomonadina, and show that „Ploeotia cf. vitrea‟ does not group with Peranemida or Petalomonadida, and thus support separateness of the three phagotrophic orders established by Cavalier-Smith (1993). Fig. 2 summarises conclusions from euglenozoan sequence phylogeny at the ordinal level based on both Hsp90 (Cavalier-Smith et al. 2016a) and the present very comprehensive site-heterogeneous 18S rDNA trees. Serpenomonas pellicle strip ultrastructure and morphogenesis differ so radically from Ploeotia and all other euglenoids, as I shall explain in detail elsewhere, that they should never have been put in the same genus (Farmer and Triemer 1988). Serpenomonas (Triemer 1986) deserves its own order Heterostavida to emphasise its radically different pellicle morphogenesis (heteromorphic strips, not homorphic ones as in Ploeotiida and most other euglenoids) and phyletic distinctiveness. All trees confirm its extreme divergence from Ploeotia cf. vitrea (Cavalier-Smith et al. 2016a). I group Heterostavida with Petalomonadida and Decastavida (recently established for Decastava and Keelungia: Cavalier-Smith et al. 2016a) as a new phagoheterotrophic euglenoid class Stavomonadea, ancestrally with a pellicle of 10 rigid simple strips and non-protrusible mouthparts, with feeding comb and two hollow support rods. The simplicity of stavomonad strips distinguishes them from Ploeotiida sensu stricto (here restricted to Ploeotia and Lentomonas) that I place in a new class Ploeotarea because of their distinctive pellicle structure and Ploeotia's deep separation from stavomonads on rDNA trees. Leedale placed Entosiphon in Heteronematina whereas Cavalier-Smith included them in Ploeotiida (1993, 1995 implicitly; explicitly in von der Heyden et al. 2004); Adl et al. (2012) left them in Heteronematina but their footnote implied a preference for Ploeotiida. Because of their inordinately long-branch for Entosiphon rDNA trees were unable to establish its correct position, but Hsp90 trees lack that long-branch problem and strongly (and site-heterogeneous rDNA trees weakly) place Entosiphon as sister to all other euglenoids (Cavalier-Smith et al. 2013a). Entosiphon does not group with either Peranema or Serpenomonas as it did inconclusively on distance rDNA trees (von der Heyden et al. 2004). This deeply branching position is consistent with its FA having three supportive rods of very distinct structure from the two present in Peranemea and Ploeotiida (Triemer and Farmer 1991a,b) and with major differences in pellicle structure. Therefore as well as removing Entosiphon from Ploeotiida as a separate order I place it in a new infraphylum and class (Table 1). That entails removing Entosiphon from Peranemea. New class Entosiphonea with protrusible siphon and three microtubule-bundle support rods emphasises that its rods differ greatly from those of other euglenoids here grouped as new infraphylum Dipilida; I consider that this structural dichotomy represents the primary phylogenetic split within euglenoids. Ribosomal DNA trees also show the original ultrastructurally diverse Peranemida to be deeply paraphyletic (Figs 1 and S2 where the former peranemids Peranema, Anisonema, Dinema, Neometanema, Teloprocta are not collectively a clade, but rhabdomonads and Euglenophyceae are independently derived from them) confirming early distance-tree evidence (von der Heyden et al. 2004), so I subdivide former Peranemida by establishing three new holophyletic orders of contrasting motility within Peranemea: Anisonemida glide on the posterior cilium not the anterior one as in Peranemida sensu stricto; Natomonadida swim instead of gliding and include ultrastructurally and phylogenetically related phagotrophs (Neometanema) as well as the osmotrophic Rhabdomonadina; Acroglissida at present includes only the anterior gliding Teloprocta scaphurum (Cavalier-Smith et al. 2016a) that uniquely for Euglenozoa has a cytoproct and was formerly wrongly treated as a Heteronema (Breglia et al. 2013). Thus there are now nine orders of non-photosynthetic euglenoids, each with distinctive ultrastructure and uniform motility mode. Each order is rather homogeneous in ultrastructure and lifestyle, whereas previously Ploeotiida and Peranemida were deeply paraphyletic and ultrastructurally extremely diverse. Eight orders are clearly clades on rDNA trees; Anisonemida is either a clade (Figs 1, S1, S2) or ancestral to the closely related Natomonadida. Sequence trees show that the difference between anterior ciliary gliding (Petalomonadida, Peranemida, Acroglissida, Postgaardida) and posterior ciliary gliding (Entosiphonida, Decastavida, Heterostavida, Ploeotiida, Anisonemida) is evolutionarily profoundly important. From their distribution on the tree we can conclude that gliding on the posterior cilium is the ancestral state for euglenoids. These contrasting locomotory patterns, which must use different molecular motors (kinesin for posterior gliding and dynein for anterior gliding: Cavalier-Smith 2013a, 2014b), appear to have been stable for hundreds of millions of years with rare switches between them, as no euglenoid orders recognised here have a mixture of anterior and posterior ciliary gliders and their divergences are all ancient. Swimming is phylogenetically much more restricted in euglenoids, characterising only two independently derived clades: Euglenophyceae and Natomonadida. Neometanema normally swim like their rhabdomonad relatives, and should never have been called Heteronema, conforming neither with Dujardin‟s original concept (posterior ciliary gliding) nor with Ritter von Stein‟s totally contradictory one (anterior ciliary gliding). The skidding mode of swimming close to surfaces of the predatory Neometanema can be regarded as the ancestral state for natomonads, evolutionarily and behaviourally intermediate between gliding and the fully planktonic swimming of rhabdomonads that presumably evolved when these osmotrophs first abandoned phagotrophy. Rhabdomonadina and Euglenophycidae were once grouped together as class Aphagea on the hypothesis of a common loss of phagotrophy and switch from gliding to swimming (Cavalier-Smith 1993). However, rDNA trees (Fig. 1) show that the ancestor of Natomonadida switched from gliding to swimming independently of Euglenophyceae and before Rhabdomonadina lost phagotrophy. As the discovery of phagophototrophic Rapaza showed (Yamaguchi et al. 2012), even Euglenophycidae did not switch to swimming and lose phagotrophy simultaneously. Swimming evolved first in the ancestor of all Euglenophyceae at the time of green algal chloroplast enslavement (Cavalier-Smith 2013b), whereas phagotrophy was lost later in only one subclade (Euglenophycidae), being retained by its sister Rapaza. Therefore I abandon polyphyletic Aphagea. Unlike Adl et al. (2005, 2012) and Lee and Simpson (2014a,b), I do not accept restricting Aphagea to Rhabdomonadina alone (Busse and Preisfeld 2003), as sequence trees favour making Anisonemia a subclass with two orders of contrasting ciliary/locomotory organisation (Table 1). Following Cavalier-Smith (1993) and contrary to Adl et al. (2005, 2012), I retain Leedale‟s suborder Rhabdomonadina (as expanded previously to include Astasia and Distigma). On its own, loss of phagotrophy is insufficient to merit even ordinal separation; it would be unwarranted rank inflation to treat both natomonad suborders as a class, which accepting class Aphagea for Rhabdomonadina would entail. Unlike Adl et al. (2005, 2012), I follow Cavalier-Smith (1993) in rejecting Leedale‟s excessively heterogeneous Heteronematida that embraced genera now included in two separate classes (Peranemea, Entosiphonea). This heterogeneity and Ritter von Stein's muddle over what Heteronema means (see Cavalier-Smith et al. 2016a) make this ordinal name best abandoned. Most described Heteronema species need to be assigned to a new genus or genera when characterized by ultrastructure and/or sequencing. Their anterior gliding and second shorter cilium suggest that most belong in Acroglissida. Only H. marina, the type, is surely a Heteronema! On Fig. 1 Peranemea and Euglenophyceae together form a very strong (0.99, 100%) clade, here made new euglenoid superclass Spirocuta on account of their spirally arranged, often contractile pellicle. The spirocute pellicle with strips interlocking laterally by complementary hooks at their heel [the name used by Mignot et al. (1987) for the thickened edge of spirocute strips clearly visble when is squashed and strips appear as separate laminae (Leedale 1996)] and opposite or 'toe' edges. This interlocking contrasts sharply with the primitive arrangement of laterally flush-butted, barrel-stave-like strips that form the rigid pellicle of the new ancestral superclass Rigimonada (Ploeotarea, Stavomonadea) as well as in Entosiphonea. Here I designate the unthickened edge of spirocute strips the toe. This new term is desirable because although Mignot at al. (1987) called the recurved edge of the spirocute toe the hook, Leander and Farmer (2001) confusingly used „the hook‟ instead for the raised edge of the heel and adopted the little-used term „overhang‟, which originally referred to the overall shape of each Euglena 'pellicle complex' (Sommer and Blum 1965) not specifically to part of a strip, changing its meaning to refer instead to the hook of Mignot et al. (1987). Retaining Mignot's older name 'heel' and adopting an unambiguous complementary new name 'toe' resolves the confusion caused by the now contradictory double meaning for 'hook' and usefully allows the same terminology for the contrasting strip edges of all euglenoids, even those of Entosiphon and rigimonads where the toe is straight, not hooked. I recommend that the now ambiguous term 'hook' be no longer used to designate specifically just one strip edge in spirocutes, both of which are hooked. Other recent rDNA trees (e.g. Lax and Simpson 2013) also show a sharp bipartition between ancestral rigid euglenoids with 12 or fewer flush-butted pellicle strips mostly with straight toes (Entosiphon plus Rigimonada) and derived classes with 16 or more spiral interlocking strips with hooked toes and widespread euglenoid motility (Spirocuta). Within Euglenophyceae adding a new phagophototrophic order for Rapaza, yields 12 euglenoid orders overall, not six as in Leedale (1967). This increase stems partly from discovery of Rapaza, Serpenomonas, and decastavids, and partly from ultrastructural and sequencing revelations of radically greater phagotrophic euglenoid diversity than appreciated in the 1960s. According to the latest sequence trees (e.g. Fig. 1 and Cavalier-Smith et al. 2016a) only one euglenoid order is undoubtedly paraphyletic (Eutreptiida); in accepting it Adl et al. (2012) did not realize its probable paraphyly, presumably because other recent sequence trees of Euglenophyceae included too few outgroups to show that (Marin et al. 2003) or none at all (Linton et al. 2010), and so may have been incorrectly rooted. Contrary to a mistaken assertion (Adl et al. 2005), creating a higher-rank taxon to include a single lower-ranked taxon is not taxonomically „superfluous‟. For example, new subclass Rapazia (with at present a single order, family and genus) allows us (a) to emphasise by its rank that the dichotomy between the photophagotroph Rapaza and the non-phagotrophic phototrophs is the most important one within Euglenophyceae; and (b) by giving equal rank to its sister subclass Euglenophycidae to indicate that Eutreptiida and Euglenida together are a clade and mutually more closely related than either is to Rapaza. The oversimplified Adl et al. (2012) system achieves neither benefit; its categorically confusing equal ranking of a single genus Rapaza, order Eutreptiales, and quasi-class „Euglenea‟ (changed in sense from 2005!) (Adl et al. 2012) is better expressed by ranking them all as orders. The standard ordinal suffix - ida automatically travels with the name, telling readers their equal rank whenever mentioned, which is not so with their cumbrous multiple blob ranking, which lacks advantages over standard, broadly understood Linnean categories like order, class, and phylum. Using blobs to denote rank, motivated primarily to avoid minor suffix changes that some rank changes entail (a non-problem) (Adl et al. 2005), did more harm than good. The square bracket system citing up to four names for each taxon (Adl et al. 2005) proposed to circumvent the harm done by unwisely abandoning rank-denoting suffixes is cumbersome in the extreme, a decisive argument against it. Taxonomy of photosynthetic non-phagotrophic euglenoids is in a relatively good state with morphological and sequence evidence well integrated and a proper Linnaean hierarchy of categories (Marin et al. 2003). That for phagotrophic and osmotrophic euglenoids has lagged considerably. That is partly because some who have been most active in studying them recently have been hostile to classical Linnean taxonomy altogether (as exemplified by Adl et al. 2005, 2012) and partly because the influential, pioneering system of Leedale (1967, 2002), despite being Linnean in character, for simplicity did not attempt to revise or even use families. The present paper is the first to include families for non-photosynthetic and phagotrophic euglenoids since Vasileva (1987) who had three phagotrophic families (Peranemataceae, Petalomonadaceae and Scytomonadaceae as in Mignot 1967), and two osmotrophic families (Astasiaceae and Menoidiaceae) for the other osmotrophs. Cavalier-Smith et al. (2016a) provided the first sequence data for a Scytomonas, showing that a separate family Petalomonadidae is unjustified. Diagnoses of the new euglenoid taxa follow in the Table 1 order. FA and pellicle structure terminology reflects the revised interpretations of homologies that I shall explain, illustrate, and reference in more detail in a separate paper.

New infraphylum Entosiphona, class Entosiphonea and Entosiphonida ord. n. Cavalier- Smith. Diagnosis: Biciliate bacterivorous euglenoids with fluted cell surface; pellicle has 4, 8, 10 or 12 longitudinal strips, whose epiplasmic layers abut laterally and do not overlap. Strip shape varies from gently undulating to markedly grooved. Feeding apparatus anteriorly with a C-shaped cement skeleton with a primary row of closely spaced embedded at its inner surface; posteriorly to this cement arc three bundles of closely packed microtubule angled rows are added to every other primary microtubule outside the now U-shaped primary row and its associated cement to make three cytopharyngeal support rods. Curved anterior supplementary plaque of cement with a thick hinged apical cap, lacking microtubules on its inner face lies inside the main U-shaped cement, thickened as a meshlike scaffold beneath the cap; these inner structures differ greatly from the broader, more massive left/dorsal jaw supports of dipilid euglenoids. Four slightly curved microtubule-attached vanes, associated with a fifth reinforced microtubule; all five microtubules loop over from a shallow side pouch of the ciliary pocket. Unlike Rigimonada, siphon protrusible and feeding comb absent. Glide on posterium cilium or swim with anterior cilium using an oar-like beat. Etymology. Named after sole included family: New family Entosiphonidae. Diagnosis: as for Entosiphona and Entosiphonida. Type genus Entosiphon Ritter von Stein, 1878. Comment: it was essential to remove Entosiphon from Anisonemidae (Lankester 1885), Peranemea (Cavalier-Smith 1993) or Ploeotiida (von der Heyden et al. 2004) into a new higher taxon, as its U-shaped FA has a more primitive character more easily related to that of diplonemids than those of dipilid euglenoids and its protrusion machinery is unrelated to that of Peranemida, as I shall explain in detail elsewhere. New infraphylum Dipilida. Diagnosis: Photosynthetic, osmotrophic or phagotrophic; if phagotrophic ancestrally with two, typically hollow cemented feeding apparatus support rods (never three), four vanes, and distinct dorsal and ventral cemented arc-like cytostomal skeletons that face each other across a ciliary pocket extension, dorsal arc wider than ventral. Etymology: di- L two-fold, double; pila L. pillar.

New superclass Rigimonada. Diagnosis: Rigid, non-squirming heterotrophic euglenoids with 10 or fewer largely longitudinal pellicular epiplasmic strips; epiplasmic strips abut laterally and do not overlap and interlock laterally as in Spirocuta; minimally two closely interlinked microtubules attached to strip heel. Ancestrally with non-protrusible feeding apparatus rods, without central microtubules or with one or a few short microtubule rows not in hexagonal array (unlike Peranemea). Rod-vane complex extends almost the whole cell length, unlike Peranemea, but cement and rods lost in petalomonads. Mostly aerobic phagotrophs. Etymology: rigeo L. I am stiff; Gk unit.

New class Stavomonadea. Diagnosis: species with cemented mouthparts have a double incurved C-shaped dense cement layer supporting its dorsal jaw, whose continuous outer lip has an associated arc of two rows of closely linked microtubules on its inner face (the outer arc with numerous projections facing the inner arc) and a third layer of widely spaced microtubules on the side facing the cytopharynx, which consists of five reinforced microtubules plus one to many pairs of unreinforced microtubules, each pair having a connecting dense lamina; the fifth reinforced microtubule is connected to the first pair by a longer lamina wrapped round a cement spur from the inner left lip. The reinforced microtubules loop over from the left/dorsal jaw support to the other side of the , where four bear vanes except in most petalomonads; the adjacent unreinforced microtubule pairs similarly loop over in parallel. FA rods hollow, with few or no internal microtubules; rods and cement lost in petalomonads. Usually glide on posterior or anterior cilium rather than swim. Strip junctions not located just below the crest of narrow prominent ridges, unlike Ploeotarea. Unlike Spirocuta and Entosiphon, cytostome separate from and ventral to reservoir canal, except in a few smaller petalomonads (Petalomonas mediocanellata, Scytomonas). Etymology: stave E. from the resemblance of the strips to barrel staves; monas Gk unit.

New subclass Homostavia. Diagnosis: 10 or fewer morphologically similar, subequal stave- like pellicular strips, lacking deep troughs, never with strong alternating heteromorphism as in Serpenomonas. Etymol: homos Gk same; stave E. because all strips have the same barrel stave, non-trough-like morphology.

New family Keelungiidae. Diagnosis: With only one pair of unreinforced microtubules looping from dorsal jaw support to cytostome; outer rod unflanged. Type genus Keelungia Chan et al., 2013. New subclass Heterostavia. Diagnosis as for sole order Heterostavida: New order Heterostavida. Diagnosis as for sole family Serpenomonadidae: New family Serpenomonadidae. Diagnosis: 10 pellicular strips symmetrically arranged but dimorphic in width and form, unlike Decastavida and Ploeotiida: five broad alternating with five very narrow and concave strips form five deep grooves underlying five, barely projecting, ridges; outermost part of ridges regularly crenate. Strip joints not on strongly projecting narrow ridges. Feeding apparatus with two dense hollow lateral rods with no internal microtubules and only a surface row facing the vanes; in posterior regions support rods have six prominent ribs (three each) associated with four unfolded vanes with one edge attached to a microtubule and two folded vanes with no edge microtubules; cytostome dorsal to anterior cilium, with prominent dorsal jaw-supporting, cemented doubly crescentic comb, its outer lip with two close- packed layers of microtubules; 12 pairs of unreinforced microtubules loop over in parallel with five reinforced microtubules from comb to cytopharynx; narrower but thicker domed ventral jaw-support connects cement rods apically. Unlike Ploeotia, there is no ventral ciliary groove delimited by two raised ridges. Type genus Serpenomonas Triemer, 1986. Etymol: hetero Gk different; stave E. because the strips are heteromorphic, only alternate ones resembling barrel staves, the others being deeply trough-like. Comment. Transferring Serpenomonas to Ploeotia (Farmer and Triemer 1988) was not justified; the asymmetric bifurcate pellicle ridges used to group them are arguably either plesiomorphies for all rigimonads or more likely convergent. rDNA trees show that Serpenomonas and P. cf. vitrea do not group together and are as far apart on the tree as any two dipilid euglenoid genera can possibly be, whereas Serpenomonas is sister to Decastavida (Fig. 1). More importantly, its strip heteromorphism is unique and not present in Ploeotia or as markedly in any other euglenoids; and it lacks Ploeotia's ventral groove. I do not accept that Serpenomonas has five vanes (Linton and Triemer 1999); as shall I explain more fully elsewhere, it has six, shown most clearly by Belhadri et al. (1992): four standard curved vanes with a microtubule along one edge, plus two additional strongly folded (one surrounding a projecting ridge from each rod) that lack edge microtubules and might arise by distal splitting of two standard microtubule-borne vanes. Linton and Triemer‟s (1999) description of the mouthparts is not fully self-consistent: the structure labeled DL in their Fig. 5 is consistent with their text „the anterior-most portion of the comb … formed what was seen externally as the dorsal lip‟, but DL in Figs 7 and 17 instead label a dorsal extension of the ventral lip cement, not the comb at all; this dorsal extension may correspond to the ventral part of the accessory lip structure present but much less prominently in Keelungia (Chan et al. 2013).

New class Ploeotarea. Diagnosis: Phagotrophic biciliate heterotrophic euglenoids; two cemented pharyngeal rods with homogeneous dense matrix, unlike Entosiphonida and Spirocuta never with hexagonal-array microtubules. 10 morphologically similar pellicular strips; joints with rounded overhangs and strongly inrolled heel region, making 10 microgrooves below the crest of 10 longitudinal ridges. Glide on posterior cilium. Etymol: ploion Gk boat, from boat- like cell shape. New family Ploeotiidae. Diagnosis: Biciliate bacterivorous rigimonads with 10 longitudinal pellicle strips. Cell in cross section rounded, with 10 narrow unequally terminally bifurcate, non-crenate ridges, each with a strip joint just below its crest. One ventral strip narrower than others forms base of a central ventral posterior ciliary groove, unlike Lentomonas. Glide on posterior cilium. Feeding apparatus with two oval hollow cement rods without obvious internal microtubules; posteriorly vanes associate with four rod cement ridges. Type genus Ploeotia Dujardin, 1841. Comment. Most nominal „Ploeotia‟ are probably wrongly in the genus as they lack 10 prominent narrow ridges. Many are more similar to Serpenomonas costata, as Lax and Simpson (2013) noted; others may not belong to either genus. From its morphology, sequenced Ploeotia cf. vitrea (Lax and Simpson 2013) probably belongs in Ploeotiidae, but is much too large to be P. vitrea; it might not even be a Ploeotia as the dorsoventral arrangement of ridges differs from P. vitrea of Farmer and Triemer (1988; not necessarily from Dujardin‟s), being more like Lentomonas. The assertion that P. vitrea and Serpenomonas costata FA are 'almost identical' was ill-documented (Farmer and Triemer 1988); no micrographs were shown of P. vitrea jaw supports, so their properties must be omitted from the diagnosis; their Fig. 9 is so fuzzy that one cannot even count vanes or decide which are folded or have edge mts, and they cannot be clearly distinguished from mt rows; there appear to be four posterior rod cement ridges not six as in Serpenomonas, but this region also differs from Lentomonas. New Family Lentomonadidae. Diagnosis: Biciliate bacterivorous rigimonads; 10 subequal pellicle strips; strip heels strongly recurved, making 10 microgrooves; dorsoventrally differentiated, three ventral strips, making flat surface, 7 dorso-lateral strips strongly curved, making 7 broad ridges, with strip joints just below prominent longitudinal ridge crests; no obvious ventral groove or ridges. Left jaw support with one arc of close-packed microtubules bearing slanted pairs of unreinforced microtubules; several widely spaced reinforced microtubules associated with ciliary pocket extension between left and right jaw supports. Cytostome separate from ciliary reservoir, no common vestibulum. Type genus Lentomonas Farmer and Triemer, 1994. Comment. L. applanatum had one narrow lateral ventral strip and three broad ones, but no ventral groove or ridges (Farmer and Triemer 1994), whereas Entosiphon applanatum (Preisig 1979) had a central narrow ventral groove bounded by two ridges. This implies a very different ventral pellicle structure, so they are not the same species. I agree with Ekebom et al. (1996) and Patterson and Simpson (1996) that ultrastructurally studied L. applanatum and Ploeotia corrugata Larsen and Patterson (1990) are the same species, and with Linton and Triemer (2001) that they are not the same genus as S. costata. I think they are also not congeneric with P. vitrea, so transfer P. corrugata and similar P. azurina to Lentomonas. New combinations: Lentomonas corrugata comb. nov. Basionym Ploeotia corrugata Larsen and Patterson, 1990 p. 867; synonym Lentomonas applanatum Farmer and Triemer (1994), but not Entosiphon applanatum Preisig, 1979. Lentomonas azurina comb. nov. Basionym Ploeotia azurina Patterson and Simpson 1996 p. 432. I based the family diagnosis entirely on Farmer and Triemer (1994), who had no light micrographs to support synonymy with E. applanatum, nor even said if FA is visible in the light microscope as in P. corrugata and E. applanatum (as Patterson and Simpson (1996) stressed, it had a protrusible siphon unlike P. corrugata).

New superclass Spirocuta. Diagnosis: Pellicle of 16-56 ancestrally spirally arranged narrow strips; ancestrally squirm by lateral sliding of strips; ancestrally phagotrophic feeders on eukaryotes with four unfolded vanes and two oval cytopharyngeal supporting rods with core of close-packed microtubules, typically in hexagonal array. Cytostome opens into upper part of reservoir (in the common vestibulum) unlike most rigimonads where it is separate from the reservoir canal as in Glycomonada. Includes secondary osmotrophs and phototrophs with reduced or absent mouthparts. Etymol: Spira L. coil, spire; cutis L. skin.

Class Peranemea Cavalier-Smith, 1993 I here emend this class by excluding Entosiphon, Serpenomonas, and Ploeotiida and by adding Rhabdomonadina, which makes it more homogeneous in pellicle and FA structure if one allows for obviously secondary FA simplifications in the osmotrophs that as losses do not deserve class-level separation. The four orders are grouped into three subclasses, each a clade on rDNA trees and each homogeneous with respect to locomotory mode and FA features. Subclass Anisonemia, the most diverse heterotrophic spirocute clade, comprises two new orders with distinct locomotory modes, each a well-supported clade. Order Anisonemida comprise biciliate phagotrophic spirocute euglenoids that invariably glide on their posterior cilium, in marked contrast to the non-sister subclasses Peranemia and Acroglissia that have a similar pellicle but glide on their anterior cilium. Anisonemida do not group with Peranema, being usually sister (Figs S1, S2 and Fig. 1 ML; but with outgroups restricted as in Fig. 1 CAT without support put them ancestral) to a consistently well supported clade of swimming heterotrophic spirocutes (new order Natomonadida) and have nonprotrusible FA. Natomonadida comprise the purely osmotrophic suborder Rhabdomonadina and new phagotrophic suborder Metanemina (Neometanema). Subclass Acroglissia (order Acroglissida) contains only the anterior ciliary glider Teloprocta scaphurum (formerly lumped in Heteronema: Skuja 1934; Breglia et al. 2013; Schroeckh et al. 2003), which unlike Peranemida sensu stricto has a non-protrusible rod apparatus and a cytoproct. Though Neometanema and Teloprocta are both phagotrophic spirocutes with simplified cytostomal supports, they never group together (despite formerly being lumped in one excessively wide genus Heteronema); as no special morphological features unite them, Cavalier-Smith et al. (2016a) made Teloprocta a separate genus. Most trees put it weakly sister to Euglenophyceae (Lax and Simpson 2013; Lee and Simpson 2014a,b; my Fig. 1); however three of my 16 best aligned and most comprehensively sampled trees group it instead with Anisonemia (Table 3), including Fig. S2 which appeared to be the most reliable for Hsp90 judging by its congruence with Hsp90, making it possible that this minority position is correct and Acroglissida plus Anisonemida are really a clade that is sister to Euglenophyceae. Highest support for an Acroglissida/Anisonemida clade (0.75) was for the largest taxon sample

(323 including Entosiphon) and largest number of included nucleotides (1577), but it was still supported (0.66) for 1577 nucleotides when Entosiphon was removed. Suppport for the competing grouping with Euglenophyceae was weak. Its position was unresolved in Breglia et al. (2013) and should become more stable when further species are sequenced. As explained by Cavalier-Smith et al. (2016a), the original Heteronema marina of Dujardin (1841) had a thicker trailing cilium and thinner undulating anterior-pointing one, thus was quite similar to his Anisonema though he put them in separate families. Ritter von Stein (1878) caused much confusion by ignoring Dujardin‟s species and applying the same generic name to two radically different euglenoids that glide instead on their anterior cilium: Peranema globulosa Dujardin, 1841 (one cilium only seen) and Astasia acus Ehrenberg, 1838. Subclass Peranemia comprises classical Peranemidae, the only family now remaining in order Peranemida (Table 1):

New subclass Peranemia. Diagnosis: Squirming phagotrophic Spirocuta that glide on anterior cilium that is rigid except for tip flickering; protrusible FA of two pharyngeal rods, with hexagonally or irregularly arranged microtubule core surrounded by cortex of microtubule-free dense cement, anteriorly closely linked by cemented connector supporting dorsal jaw; additional microtubules face vanes and sometimes (Urceolus) also surround cortex as a thin 1-mt thick peripheral „skin‟; rods each anteriorly with a long oblique, robust, lateral cemented strut (inner and outer laminas) linked by long striated fibres to each end of outer part of strongly cemented double dorsal/left jaw support (LJS) and in this region only an outer groove. LJS with lateral dense bodies; outer one linked by cemented rod-like anchor to reservoir canal peripheral cement support. Jaw supports separated at outer edge and centrally by a ciliary pocket extension across which reinforced microtubules and a row of unreinforced microtubule pairs loop from LJS to cytopharynx. Four slightly curved (Peranema) or folded (Urceolus) vanes edged by reinforced microtubules. Posterior cilium not visible by LM: absent (Urceolus, Jenningsia) or attached laterally to body within specialized groove, so invisible in LM (Peranema).

New subclass Anisonemia. Diagnosis: Ancestrally biciliate squirming non-photosynthetic spirocutes; FA of phagotrophs with two anteriorly linked rods of hexagonally close packed microtubules, peripheral ones embedded in variably developed dense cement, without a thick microtubule-free cortex (unlike Peranemia); non-protrusible, inner and outer laminas and linked striated fibres absent or vestigial. LJS often less strongly cemented than in Peranemia, without cement anchor to canal cement; rods and vanes lost by non-phagotrophic Rhabdomonadina, but reservoir canal peripheral fibrous supports well developed. Posterior cilium lost by Astasiidae.

New order Anisonemida. Diagnosis: Typically non-swimming phagotrophic biciliates with spiral pellicular strips and lashing anterior cilium that glide on prominently thickened posterior cilium. Etymol: aniso Gk unequal; nema thread, because of unequal ciliary morphology and function and because it includes Anisonema.

New order Natomonadida. Diagnosis: Heterotrophic swimming, non-gliding, biciliates with spiral pellicular strips. Apical end of vestibulum surrounded by a scroll-like structure consisting of microtubules and dense fibrous elements. Etymol: nato L. I swim, monas Gk unit. New suborder Metanemina. Diagnosis: phagoheterotrophic spirocutes with two swimming cilia of equal thickness. Non-gliding skidding motility close to surfaces driven by curved anterior-directed cilium. Etymol: meta Gk after, nema Gk thread. Non-typified name. New family Neometanemidae. Diagnosis: flattened broadly ellipsoidal biciliate phagotrophs that swim, skidding close to substrates, but do not normally glide on either cilium; cilia of equal thickness and length, anterior lashing, posterior trailing. Feeding apparatus visible or not by light microscopy. Weak to moderate squirming. Type genus Neometanema Lee and Simpson, 2014.

New subclass Acroglissia and new order Acroglissida. Diagnosis: biciliate phagoheterotrophic spirocutes; glide on proximally rigid anterior-directed cilium; weak to moderate squirming; two non-protrusible FA rods visible in light microscope. Etymol: acro Gk topmost, glisser Fr. slide, as the gliding cilium projects forwards from the cell tip. Sole genus Teloprocta Cavalier-Smith in Cavalier-Smith et al., 2016a.

Historical Note: Separation here of heterotrophic euglenoids into four ultrastructurally and phyletically distinct classes shows how much wiser Lankester (1885) was in separating them into four separate families than was Klebs (1892) who lumped them into just one, an oversimplification that so many since, up to and including Adl et al. (2005, 2012), have essentially followed. Allowing for genera then unknown, Peranemidae is compositionally identical in Lankester's and Table 1; his Petalomonadidae, a junior synonym by a year of Scytomonadidae, is also compositionally homogeneous, but unlike Scytomonadidae now excluded the biciliate Tropidoscyphus. His other two families are more heterogeneous, but before electron microscopy the FA contrasts of Entosiphon and Anisomema could hardly have been forseen; nor could those amongst genera in his Astasiidae; his Menoidina is equivalent to Rhabdomonadina except for excluding Astasia.

Class Euglenophyceae - the only algal class of Euglenozoa: chloroplast with triple envelope

New subclass Rapazia and new order Rapazida. Diagnosis: phagotrophic photosynthetic eukaryovorous euglenoids with no rod/vane feeding apparatus; simple reservoir-associated feeding pocket supported by a bundle of four rows of close-spaced microtubules, not just one row as in Euglenophycidae; non-gliding swimmers in the water column. Etymol: rapax L. seizing, grasping, as the only predatory Euglenophyceae. Comment: I consider that homologies of the Rapaza FA and the MTR pockets of Euglenophycidae (Shin et al. 2002) compared with other Euglenozoa were previously partially misinterpreted and will present a new synthesis elsewhere. New family Rapazidae. Diagnosis: as for Rapazida, plus pellicle of 16 strips and one posterior whorl, and feed on eukaryote algae. Type genus Rapaza Yamaguchi et al. (2012).

New subphylum Postgaardia Reconstructions of FA ultrastructure in Postgaardi (Simpson et al. 1996/7; Yubuki et al. 2013) and Calkinsia (Yubuki et al. 2009) confirmed that they are fundamentally similar and deserve to be classified together as a distinct order Postgaardida (Cavalier-Smith 2003a) and class Postgaardea (Cavalier-Smith 1998), as both genera uniquely share six finger-like projections inside the cytopharynx mouth with identical underlying fibrous and microtubular skeleton. Cavalier-Smith (2003a,b) had formally placed Calkinsia within Postgaardea and Postgaardida, but Yubuki et al. (2009) did not accept that these two genera were related and proposed a new clade name Symbiontida for Calkinsia plus unspecified related anaerobic Euglenozoa with epibiotic bacteria and some environmental rDNA sequences; oddly they did not include Postgaardi in Symbiontida, but confusingly incorrectly regarded the whole class Postgaardea as a synonym for the species Postgaardi mariagerensis alone. Breglia et al. (2010) discovered a substantially different third postgaardean genus Bihospites whose FA can be more readily homologised with that of diplonemids than with that of euglenoids, contrary to their interpretations (Cavalier-Smith unpublished). Yubuki et al. (2013) conjectured that the environmental DNA clade that is sister to Calkinsia in Fig. 1 is Postgaardi, thereby (like Adl et al. 2012) accepting that Postgaardi belongs in the same clade as Calkinsia and Bihospites, for which the oldest name is Postgaardea, but overlooked that Cavalier-Smith (2003a,b) had already placed Calkinsia in Postgaardea and order Postgaardida; their Fig. 24 effectively made the clade name Symbiontida a junior synonym of both Postgaardea and Postgaardida. Initially I intended to adopt Symbiontida as the subphylum name, to validate it as a ranked taxon and thereby facilitate its continued use even though when first published I considered it superfluous, but a referee requested that I use a new name instead. Breglia et al. (2010) mistakenly considered the radically novel „rod apparatus‟ of Bihospites as homologues of euglenoid rods and overlooked their greater ultrastructural similarities with diplonemid FA structures; I explain elsewhere my view that they represent hypertrophied diplonemid-like ultrastructural features (Cavalier-Smith unpublished). The postgaardean FA is not specifically euglenoid in character and I consider that the S-shaped pellicle units of Bihospites were misinterpreted as euglenoid-like. Here I establish a new postgaardean order Bihospitida for these highly distinctive flagellates, which makes Symbiontida a junior synonym only of Postgaardea. Presumably partly because of this ultrastructural misinterpretation and partly because of poorly sampled site-homogeneous 18S rDNA trees that sometimes put postgaardeans weakly within euglenoids (e.g.Yamaguchi et al. 2012; Yubuki et al. 2009), Lax and Simpson (2013), Lee and Simpson (2014a,b), and Chan et al. (2015) all controversially treat Postgaardea as euglenoids and do not accept a separate class. However, my comprehensive site-heterogeneous trees all exclude Postgaardea/Symbiontida from Euglenoida (robustly in Figs 1, S1 more weakly in Fig. S2), as do all but one of my ML trees, and weakly suggest that Postgaardea are sisters of Euglenoida, not Glycomonada. Thus their insignificantly supported intrusion into euglenoids in Yubuki et al. (2009), Yamaguchi et al. (2012), and Lax and Simpson (2013) are most likely random artifacts of including too few nucleotide positions. Two other even less well resolved trees were more grossly misleading: in Breglia et al. (2010) diplonemids, kinetoplastids, and postgaardids all wrongly nested within euglenoids, grouping with spirocutes as a collapsed tetrafurcation, whereas in Chan et al. (2013) (whose alignment was very inaccurate) both diplonemids and postgaardids were wrongly within euglenoids. Trees of Lee and Simpson (2014a,b) without glycomonads or non-euglenozoan outgroups were arbitrarily rooted to make it wrongly appear that postgaardids are nested within euglenoids. Adl et al. (2012) more reasonably treated Symbiontida as a fourth euglenozoan group of equal rank with Euglenoida (unwisely called Euglenida: see above), Diplonemea, and Kinetoplastea. Given my much more robust and consistent rDNA trees and reinterpretation of their ultrastructural homologies, I still firmly exclude Postgaardea from Euglenoida, and rank postgaardeans (= Symbiontida), Euglenoida, and Glycomonada, which collectively embrace all euglenozoan diversity, as subphyla in my revised higher classification of Euglenozoa (Table 1). This ranks equally the three deepest branching euglenozoan clades, whose exact branching order is still uncertain (Figs 1, S1, S2); each has radically distinct variants of the basic euglenozoan body plan.

New subphylum Postgaardia. Diagnosis: Biciliate free-living anaerobes covered with epibiotic bacteria in longitudinal rows. Highly contractile pellicle underlain by numerous equally spaced microtubules without specially differentiated morphogenetic pairs. Simplified cytopharynx without cytostomal or reservoir encircling fibres, cemented jaw supports or rigid longitudinal straight cemented rods. Etymology: based on on sole included class Postgaardea. Class Postgaardea Cavalier-Smith, 1998. Revised diagnosis: Biciliate heterotrophic anaerobes with well developed heteromorphic latticed paraxonemal rods and rows of epibiotic bacteria, and simplified FA. Centrioles parallel, connected by two dissimilar striated roots, thinner on cell's right associated with the intermediate centriolar root; subapical. Cytostome dorsal, not ventral as in euglenoids. Corset of longitudinal pellicle microtubules, evenly and closely spaced with frequent cross linkers and direct links to plasma membrane, apart from interruption by tubular extrusome docking sites; extending over the whole cell surface a short way into the neck (canal) of the reservoir as a dorsal row of microtubules linked to the dorsal centriolar root by a dense dorsal amorphous fibre unlike euglenoids without discrete longitudinal epiplasmic strips. Mitochondria without obvious cristae or kinetoplasts. Order Postgaardida Cavalier-Smith, 2003. Revised diagnosis: Microtubule band of four or about sixteen microtubules loops over from ciliary pocket to cytopharynx dorsal margin in a plane orthogonal to five reinforced microtubules that loop over apically from ciliary pocket to cytopharynx via six finger-like projections inside mouth of cytopharynx; no supporting rods or dorsal/left jaw support homologues. Rigid cells with homogeneous pellicle with a dense thin (~25 nm) lamina underlying microtubules; cytostome ventral groove. New family Calkinsiidae. Diagnosis: Phagotrotrophs with MTR pocket cytopharynx that eat and bacteria; cytostome opens on right of dorsal cilium into a shared apical depression (vestibulum) close to ciliary pocket; the cytopharyngeal loop has about 16 microtubules with dense flanges on the inner side of the loop at the cytopharyngeal end. Glide on largely rigid anterior cilium. Type and sole genus Calkinsia Lackey, 1960. New family Postgaardidae. Diagnosis: with cytopharynx opening separately from ciliary pocket; 5 reinforced microtubules (MTR) loop from ciliary pocket, along right side of U-shaped gutter oriented posteriorly from the cytostome, turn anteriorly along its left side halfway back towards cilia, then loop inwards and posteriorly alongside cytopharynx that opens into left gutter; an orthogonal looping band of four non-flanged microtubules passes from ciliary pocket end of MTR along the anterior half of the left gutter; gutter covered by two overlapping longitudinal flaps, inner reinforced ridge on left and anterior lip on right. Swim, not glide. Type and sole genus Postgaardi Fenchel et al., 1995.

New order Bihospitida. Diagnosis: as for new family Bihospitidae. Diagnosis: Metabolic anaerobic bacterivorous phagotrophs with pellicle longitudinally subdivided into extrusome- delimited S-shaped regions formed by grooves containing epibiotic bacteria and ridges with underlying mitochondria; cytostome opens on right of dorsal cilium into shared apical depression (vestibulum) close to ciliary pocket. Strongly curved C-shaped rod apparatus originates at vestibulum, loops round nucleus within a nuclear envelope groove; nucleus- attached „main rod‟ comprises a dense lamina (immediately adjacent to adhering accessory rod) bearing a stack of ~75 broad, parallel, laminas; „accessory rod‟ is a membrane-associated row of ~40 reinforced microtubules with proximal dense flanges and distal paired vanes like those of diplonemids, initiated at a cytostomal funnel at the ciliary pocket and looping over to cytopharynx; an adjacent bundle of non-reinforced microtubule pairs at the ciliary pocket is not part of the 'accessory rod'. Type genus Bihospites Breglia et al., 2010.

Subphyla Plicostoma and Saccostoma abandoned Larsen and Patterson (1990) grouped diplonemids and euglenoids as 'plicostome Euglenozoa' because they considered the 'plicate' (folded) vanes of , Ploeotia and Serpenomonas mouthparts homologous and a synapomorphy absent in kinetoplastids. Cavalier- Smith (1998) formalised this group as subphylum Plicostoma and erected a complementary subphylum Saccostoma for Kinetoplastea and Postgaardea, assuming that their simpler mouthparts without vanes were primitive. The assumptions behind these proposals now appear to be erroneous, as I argue elsewhere that the four euglenoid vanes were not ancestrally plicate and that diplonemids do not have five vanes, three plicate, as originally assumed, but eight vanes of which six are paired, these pairs having been misinterpreted as folded vanes. Furthermore, the previously misinterpreted 'accessory rod' of the postgaardean Bihospites is probably an 8-fold multiplication of diplonemid-like paired vanes, implying that such paired (not-plicate) vanes were ancestral to all Euglenozoa (Cavalier-Smith unpublished). Though Plicostoma (euglenoids plus Diplonemea) was a weakly supported clade on many 18S rDNA trees (e.g. von der Heyden et al. 2004), phylogenetic trees for several proteins showed this group to be paraphyletic; instead classes Kinetoplastea (trypanosomatids and their free-living bodonid and prokinetoplastid relatives) and Diplonemea were sister groups (Simpson et al. 2006; Simpson and Roger 2004). Recent multigene trees strongly support holophyly of the Diplonemea/Kinetoplastea clade (Cavalier-Smith et al. 2014, 2015a,b, 2016b), which is also now consistently strongly supported by the most comprehensive site-heterogeneous 18S rDNA trees published here as well as by Hsp90 trees (Cavalier-Smith et al. 2016a). I call the Diplonemea/Kinetoplastea clade Glycomonada because uniquely among eukaryotes peroxisomes were modified in their last common ancestor to form glycosomes containing glycolytic enzymes that are present only in the cytosol in other lineages (Makiuchi et al. 2011; Gualdrón-López et al. 2012). The seemingly simple vaneless mouthparts of Postgaardi and most kinetoplastids are almost certainly independent simplifications of a vaned ancestor, analogous to those that occurred independently in Euglenophycidae and most petalomonads (Cavalier-Smith et al. 2016a). Fig. 1, S1, S2 robustly confirm that Plicostoma are paraphyletic and provide the first rooted euglenozoan trees to unambiguously show that Saccostoma is polyphyletic; as both were also based on ultrastructural/evolutionary misinterpretations, they are discontinued as taxa [in agreement with Ruggiero et al. (2015)]. As diplonemids and kinetoplastids clearly have the same fundamental body plan, I make Glycomonada a new subphylum of Euglenozoa:

New subphylum Glycomonada. Diagnosis: Heterotrophic Euglenozoa without pellicular strips; microbodies are glycosomes containing glycolytic enzymes, not peroxisomes. Mitochondria ancestrally polykinetoplastid; mitochondrial genomes of multiple heterogeneous circles; transcripts undergo RNA editing including uridine insertion. Ciliary pocket without dorsal row microtubules, unlike Euglenoida; pellicle microtubules ancestrally in a continuous cross-linked corset, nucleated posteriorly; cytostome ancestrally present at tip of pronounced apical rostrum separated from ciliary pocket by narrow preoral crest; feeding apparatus ancestrally with left and right 'jaw-bone'-like cement lip supports, each associated with short lateral dense rods that extend only a short way from cytostome; ancestrally with hairs on preoral crest, and circumferential encircling microtubules surrounding cytostome; three microtubule sets (nucleated in ciliary pocket) loop over to support the left side of cytopharnx: 4-8 central widely spaced microtubules reinforced by characteristic dense material (MTR) flanked by close-set less reinforced microtubules on dorsal (parallel microtubule loop (PML) microtubules, mostly paired with intrapair thin laminas) and ventral (external microtubule band (EMB) sides; ancestrally MTR microtubules had flanges on inner face of loop and vanes on cytostome- associated outer part, secondarily lost in most kinetoplastids. Etym: glycys Gk sweet, monas Gk unit, because of glycosomes. Comment: 187-8-gene trees maximally support (by CAT and ML) Diplonemea plus Kinetoplastea being a clade (Cavalier-Smith et al. 2014, 2015a,b, 2016b), and they share a unique mitochondrial genome structure of multiple heterogeneous circles (Marande et al. 2005; Roy et al. 2007). Though mitochondrial genomes of euglenoids are also peculiar and poorly characterised compared with non-Euglenozoa, in Petalomonas cantuscygni at least they appear predominantly linear (Roy et al. 2007). mitochondrial genomes comprise a pool of heterogeneous DNA molecules encoding fewer proteins than the 12 in kinetoplastids - only seven, whose transcripts do not undergo RNA editing (Dobáková et al. 2015). RNA editing by uridine insertional and/or deletion is therefore a derived shared character of glycomonads, not ancestral for Euglenozoa whose last common ancestor clearly reduced mitochondrially encoded protein numbers compared with excavate protozoa, but retained 43 proteins from their bacterial ancestors that were lost by excavates and their higher eukarote descendants. Cytopharynx and associated mts lost by some trypanosomatids.

Class Diplonemea, order Diplonemida Montegut-Felkner and Triemer (1996) made the first three dimensional reconstruction of a diplonemid FA. Until recently the taxonomic position of Hemistasia was unknown. Initially considered a (Scherfell 1900) or euglenoid relative (Griessmann 1913), ultrastructure showed many similarities to kinetoplastids (Elbrächter et al. 1996), as Senn (1911) supposed. Simpson (1997) suspected it was a diplonemid; Yabuki and Tame (2013) confirmed this by rDNA sequencing and suggested it merits a new family. I now establish new family Hemistasiidae in order Diplonemida. Hemistasia FA resembles that of Diplonemidae more than previously realised; elesewhere I use comparisons between them to reconstruct the ancestral diplonemid FA. Similarities between the of the now broadened Diplonemea and Kinetoplastea are much greater than between diplonemids and euglenoids. New family Hemistasiidae. Diagnosis: phagotrophic biciliates with obvious rostrum and apical cytopharynx with supporting microtubules and two hollow dense cement rods; differ from Diplonemidae in having mitochondria with giant flat cristae and dispersed kinetoplast nodules and two long cilia in trophic cells, ciliary pocket extension very shallow, and having smooth cortical alveoli. Paraxonemal rods and tubular extrusomes prominent. Peripheral lacunae in cytoplasm. Type genus Hemistasia Griessman (1913). Comment: Transferring Phyllomitus amylophagus to Hemistasia (Lee 2002) was wrong if Mylnikov et al. (1998) correctly identified their strain that did not have cortical alveoli or a well developed set of encircling microtubules; this species is probably a neobodonid deserving a new genus; I agree with Lee (2002) that it is not Phyllomitus and that genus must be restricted to marine P. undulans Stein. As Phyllomitus is unassigned to a suprageneric taxon I now formally classify it within phylum , Class , order Marimonadida that contains the only other known genera with mutually adhering posterior-pointing cilia: Auranticordis with four; Rhabdamoeba with two as in Phyllomitus (Howe et al. 2011). Abollifer with parallel centrioles and two non-adhering cilia shown to be a marimonad by sequencing (Shiratori et al. 2014) has a similar apical ventral pit or short groove to Phyllomitus and likewise is highly flexible. Pseudophyllomitus with four species, perhaps not a coherent genus (Lee 2002), might in part belong to diplonemids or (more likely) Cercozoa or elsewhere, but must be left incertae sedis in no phylum. „Phyllomitus apiculatus‟ ultrastructurally studied by Mylnikov (1986) was misidentified and probably an undescribed relative of Rhynchobodo.

Class Kinetoplastea: three new bodonid families Multigene trees strongly confirm that Prokinetoplastina and Metakinetoplastina are deeply divergent sister clades (Cavalier-Smith et al. 2016b). Bodonida, though paraphyletic (Cavalier-Smith et al. 2014, 2015a,b, 2016b; Deschamps et al. 2010), are kept as an order, neobodonids, parabodonids, and eubodonids being reduced to suborders as their phenotypic differences are too slight to merit the ordinal rank assigned in Moreira et al. (2004). My site- heterogeneous trees (e.g. Fig. 1, S1, S2) strongly confirm the holophyly of Parabodonina and Eubodonina shown originally by site-homogeneous trees (Moreira et al. 2004; von der Heyden et al. 2004), but are inconclusive over whether Neobodonina are paraphyletic [ancestral metakinetoplastids; also seen (statistically insignificantly) on a recent neighbor joining tree (Hirose et al. 2012)] or a sister clade to all other metakinetoplastids. As no families exist for neobodonids and the nine genera fall naturally into two groups with substantially distinct ultrastructure, I establish two neobodonid families, Rhynchomonadidae being a maximally supported clade on all my trees. Neobodonidae is paraphyletic like itself and Neobodo designis that is so deeply diverse it needs thorough study to make numerous separate species (von der Heyden and Cavalier-Smith 2005). Parabodo can no longer remain in Bodonidae; I could have simply transferred it to Cryptobiidae, but it is sufficiently different to merit its own family; is paraphyletic and needs revision. These three new families correct the irrational situation prevailing since Moreira et al. (2004) of these 10 genera remaining in family Bodonidae with this family spread across three separate orders! Formerly a catch-all, Bodonidae is now homogeneous and restricted to Bodo for the first time in over 130 years since it was established in 1883. New family Neobodonidae. Diagnosis: biciliate kinetoplastids with apical cytostome and gently curved longitudinal cytopharynx occupying half to two thirds cell length, substantially occupying a rigid rostrum on the same side as the anterior undulating cilium; mouthparts only slightly retractile. Posterior cilium trails behind during swimming. Intermediate root of several microtubules. Cross-linked pellicle microtubules evenly spread under surface. Cytostome supported on cilium side by five or more widely spaced microtubules, with dense reinforcing matrix and V- or Y-shaped dense linkers to plasma membrane, that loop over from ciliary pocket in parallel with a 2-3 mt band of closely interlinked microtubules, and by a prism or trapezoidal rod organ of 2-6 microtubule rows, apically fixed to MTR/cytopharynx complex by dense amorphous cement, C-shaped in at least some species; outer cytostome lip supported by curved microtubule band. Phagotrophic; bacterivores or eukaryovorous. Type genus Neobodo Vickerman in Moreira et al., 2004. New family Rhynchomonadidae. Diagnosis: the non-swimming anterior cilium adheres to the mobile proboscis, swinging it from side to side as it beats; this, plus gliding on posterior cilium, and left jaw support being a single microtubule row, without rod organ, distinguish them from Neobodonidae. Five MTRs and two or three external microtubule band microtubules border cytopharynx; separate arc-like microtubule bands at proboscis base and tip. Type genus Rhynchomonas Klebs, 1892. New family Parabodonidae. Diagnosis: biciliates with anterior swimming cilium and posterior trailing one, without proboscis or rostrum. Trailing cilium attached basally at least to cell body. Cytopharynx straight, shorter than in neobodonids, obliquely positioned with cytostome behind trailing cilium; supported by MTR/AL and 2-mt parallel microtubule loop; microtubular rod organ absent or reduced to 5-mt vestige or single row. Intermediate centriolar root of one microtubule. Outer cytostome lip‟s curved microtubular supporting band with numerous or as few as two microtubules. Non-gliding, bacterivorous phagotrophs or osmotrophs. Type and sole genus Parabodo Vickerman in Moreira et al. (2004).

Euglenozoan ciliary functional divergence My new classification segregated the posterior-gliding Anisonemidae as a new order Anisonemida and grouped all swimming plastidless euglenoids (Neometanema and the osmotrophic Rhabdomonadina) as Natomonadida. Anterior-gliding Teloprocta is separated from Peranemida as order Acroglissida. Thus Peranemida sensu stricto now has only Peranemidae (four genera). All these orders of Peranemea are ultrastructurally/behaviourally uniform but distinct from the other three, as well as phylogenetically so deeply divergent that their distinctive ultrastructure and ciliary locomotory patterns must be very ancient, dating back at least to the Palaeozoic. Ciliary gliding may have played a key role in the origin of cilia in the ancestral eukaryote as an intermediate stage in evolution prior to the mechanistically more complicated ciliary swimming (Cavalier-Smith 2014b). According to that scenario the last common ancestor of all eukaryotes moved by kinesin-driven posterior ciliary gliding and fed by fishing for bacteria with an undulating anterior cilium that used surface gliding motility to move trapped bacteria to the cell surface [as is done today in the amoebozoan filosum (Smirnov et al. 2011) and the euglenoid Scytomonas saepesedens, and possibly Teloprocta scaphurum

(Breglia et al. 2013)], but had no discrete cytopharnyx nor specialized ventral feeding groove. It was further argued that following a putatively basal eukaryote bifurcation between neokaryotes and Euglenozoa (Cavalier-Smith 2010a,b), excavates (the ancestral neokaryotes) evolved planktonic swimming and a ventral feeding groove and lost gliding motility, whereas Euglenozoa evolved a cytopharynx and retained a benthic or surface-associated life style retaining ciliary gliding. The ancestral euglenozoan might have inherited posterior ciliary gliding directly from the ancestral eukaryote, it being lost by ancestral neokaryotes when feeding grooves evolved. If the basal euglenozoan bifurcation is between glycomonads, in which anterior gliding is unknown but posterior gliding present in some neobodonids, and euglenoids/postgaardids, where Calkinsia and three euglenoid orders have anterior ciliary gliding and five euglenoid orders have posterior ciliary gliding, anterior gliding probably evolved four times independently in Euglenozoa. In other also posterior gliding is much more widespread, being the predominant motility mode in Sulcozoa and Cercozoa and present in one genus (Caecitellus). In Sulcozoa their ancestral posterior gliding was replaced by anterior gliding when breviates lost the posterior cilium (Cavalier-Smith 2013), and in Cercozoa anterior gliding evolved in skiomonads, which alone among eukaryotes glide on both cilia (Cavalier-Smith and Karpov 2012; Howe et al. 2011a). In Chlamydomonas has bidirectional intraciliary motility in both cilia mediated by intraciliary transport particle trains driven one way by dynein and the other by kinesin motors (Shih et al. 2013), their dynein 1b causing cell gliding with anterior-directed cilium. All ciliated eukaryotes use kinesin-based anterograde transport for ciliary growth (Scholey 2013a, b) and I suspect dynein-based retrograde transport for retraction (Dentler 2005). If, as is likely, all ciliated eukaryotes have intraciliary transport particle trains driven by both dynein and kinesin, the direction of ciliary gliding on substrata would depend on which of these motors is activated when coupled to surface glycoproteins (Shih et al. 2013) rather than on their presence or absence. The more even spread of anterior and posterior gliding in Euglenozoa might be attributable solely to differential loss from a common ancestor that had both, but the fact that no modern euglenoids have retained both and that only two eukaryote genera (the skiomonads Tremula and Glissandra (Howe et al. 2011)) glide on both cilia suggests that biciliary gliders occupy a very narrow zone and that biciliary gliding was probably an evolutionarily unstable state for euglenoids, though it might have existed temporarily during the switches from one mode to another when orders diverged. If all euglenoid anterior gliding evolved secondarily, as in Sulcozoa and Cercozoa, intermediates where switching occurred could have been dual gliders like skiomonads, but perhaps more likely near-surface skidding swimmers like Neometanema, an evolutionary stable adaptive zone for euglenoids; loss of posterior gliding coupled with retention of surface-hugging swimming behaviour could have preadapted them for secondary evolution of anterior gliding, simply explaining why it is commoner in euglenoids than other phyla.

Derived nature of petalomonad feeding mode The common idea that the first euglenoids were strict bacterivores (Leander 2004; Triemer and Farmer 1991a,b) is ecologically and evolutionarily unrealistic, and the associated idea that petalomonad simple FA is primitive is contradicted by my improved rDNA trees showing petalomonads as nested within lineages with highly complex FA and the discovery of more complex FA in Scytomonas than other studied petalomonads (Cavalier-Smith et al. 2016a). More likely ancestral euglenoids opportunistically fed on both bacteria and protists, and were of moderate, not tiny, size. Ability of the 54 m „Ploeotia aff. vitrea‟ to eat algae (Lax and Simpson 2013) and the phylogenetically distant Serpenomonas to eat yeast (Linton and Triemer 1999) show that stavomonad rigid pellicles are not barriers to eukaryovory, as often assumed (Triemer and Farmer 1991b); small size, mouthpart simplification, or phylogenetic specialization in food preference better explain why stavomonads are mostly bacterivorous. Most are so little studied that our view of their diet may be biased; eukaryovores are harder to cultivate than bacterivores. Even Scytomonas ingests quite large cells (Cavalier-Smith et al. 2016a) and could in principle have enslaved a cyanobacterium, as did and the ancestor of plants (Cavalier-Smith 2013b). However, if euglenoids are ~1.2 Gy old but only ~750 My old (see Cavalier-Smith 2013a), for much of their early history stavomonads existed in a world with no green or other eukaryotic algae to eat. According to Fig. 1 Euglenophyceae are roughly half as old as Euglenozoa, and Euglenophyceae only about three quarters as old as Spirocuta, so Spirocuta probably evolved only after eukaryotic algae, whose origin would have given a selective advantage to greater cell size via strip multiplication. Perhaps, the classical view should be turned on its head; eukaryovory may be the ancestral condition for Euglenozoa and may have stimulated the origin of the FA. Some postgaardeans, diplonemids and kinetoplastids are eukaryovores, so why should we suppose that all early euglenoids ate only bacteria?

Concluding Comment In my view this revised classification better partitions the immense cellular diversity of phagotrophic euglenoids that is now increasingly apparent as well as the fundamental ultrastructural differences amongst the three subphyla than do any previous classifications of

Euglenozoa. However, as Euglenozoa of interestingly distinct ultrastructure will probably continue to be discovered for at least another decade, it is likely to need some further revision. Nonetheless I hope its main features will stand the test of time and contribute to greater taxonomic stability in the future.

Acknowledgements I thank NERC for past grant and Professorial Fellowship support and funding open access and Ya-Fan Chan for kindly providing his sequence alignment.

References

Adl, S.M., Simpson, A.G., Farmer, M.A., Andersen, R.A., Anderson, O.R., Barta, J.R., Bowser, S.S., Brugerolle, G., Fensome, R.A., Fredericq, S., James, T.Y., Karpov, S., Kugrens, P., Krug, J., Lane, C.E., Lewis, L.A., Lodge, J., Lynn, D.H., Mann, D.G., McCourt, R.M., Mendoza, L., Moestrup, O., Mozley-Standridge, S.E., Nerad, T.A., Shearer, C.A., Smirnov, A.V., Spiegel, F.W., Taylor, M.F., 2005. The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J. Eukaryot. Microbiol. 52, 399-451. Adl, S.M., Simpson, A.G., Lane, C.E., Lukeš, J., Bass, D., Bowser, S.S., Brown, M.W., Burki, F., Dunthorn, M., Hampl, V., Heiss, A., Hoppenrath, M., Lara, E., Le Gall, L., Lynn, D.H., McManus, H., Mitchell, E.A., Mozley-Stanridge, S.E., Parfrey, L.W., Pawlowski, J., Rueckert, S., Shadwick, R.S., Schoch, C.L., Smirnov, A., Spiegel, F.W., 2012. The revised classification of eukaryotes. J. Eukaryot. Microbiol. 59, 429-493. Allen, J.W.A., 2011. Cytochrome c biogenesis in mitochondria - systems III and V. FEBS J. 278, 4198- 4216. Belhadri, A., Brugerolle, G., 1992. Morphogenesis and ultrastructure of the feeding apparatus of Entosiphon sulcatum: an immunofluoresence and ultrastructural study. Protoplasma 168, 125- 135. Belhadri, A., Bayle, D., Brugerolle, G., 1992. Biochemical and immunological characterization of intermicrotubular cement in the feeding apparatus of phagotrophic euglenoids: Entosiphon, Peranema, and Ploeotia. Protoplasma 168, 113-124. Bicudo, C. E. de M., Menezes, M., 2016. Phylogeny and classification of Euglenophyceae: a brief review. Frontiers Ecol. Evol. 4, article 17. doi: 10.3389/fevo.2016.00017 Blochmann, F., 1895. Die microscopische Pflanzen-und Thierwelt des Susswassers. Theil II. Abteilung I: Protozoa. Lucas Gräfe & Sillem, Hamburg. Breglia, S.A., Yubuki, N., Hoppenrath, M., Leander, B.S., 2010. Ultrastructure and molecular phylogenetic position of a novel euglenozoan with extrusive episymbiotic bacteria: Bihospites bacati n. gen. et sp. (Symbiontida). BMC Microbiol. 10, 145. Breglia, S.A., Yubuki, N., Leander, B.S., 2013. Ultrastructure and molecular phylogenetic position of Heteronema scaphurum: a eukaryovorous euglenid with a cytoproct. J. Eukaryot. Microbiol. 60, 107-120. Brown, M.W., Sharpe, S.C., Silberman, J.D., Heiss, A.A., Lang, B.F., Simpson, A.G., Roger, A.J., 2013. Phylogenomics demonstrates that breviate flagellates are related to opisthokonts and apusomonads. Proc. R. Soc. Biol. Sci. 280, 20131755.

Brugerolle, G., 1985. Des trichocystes chez les bodonides, un caractère phylogénétique supplémentaire entre et Euglenida. Protistologica 21, 339-348. Burki, F., Kaplan, M., Tikhonenkov, D.V., Zlatogursky, V., Minh, B.Q., Radaykina, L.V., Smirnov, A., Mylnikov, A.P., Keeling, P.J., 2016. Untangling the early diversification of eukaryotes: a phylogenomic study of the evolutionary origins of Centrohelida, Haptophyta and . Proc. Roy. Scoc. Biol. Sci. 283. Busse, I., Preisfeld, A., 2003. Systematics of primary osmotrophic : a molecular approach to the phylogeny of Distigma and Astasia (Euglenozoa). Int. J. Syst. Evol. Microbiol. 53, 617-624. Bütschli, O., 1884. Dr. H. G. Bronn's Klassen und Ordnungen des Thier-Reichs. Vol. 1 Abt. II Mastigophora C. F. Winter, Heidelberg. Calkins, G.N., 1926. The Protozoa. Lee and Fibiger, New York. Cavalier-Smith, T., 1978. The evolutionary origin and phylogeny of microtubules, mitotic spindles and eukaryote flagella. BioSystems 10, 93-114. Cavalier-Smith, T., 1981. Eukaryote kingdoms: seven or nine? BioSystems 14, 461-481. Cavalier-Smith, T., 1993. Kingdom Protozoa and its 18 phyla. Microbiol. Rev. 57, 953-994. Cavalier-Smith, T., 1995. Zooflagellate phylogeny and classification. Tsitologiia 37, 1010-1029. Cavalier-Smith, T., 1998. A revised six-kingdom system of life. Biol. Rev. Camb. Philos. Soc. 73, 203-266. Cavalier-Smith, T., 2002. The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa. Int. J. Syst. Evol. Microbiol. 52, 297-354. Cavalier-Smith, T., 2003a. The excavate protozoan phyla Metamonada Grassé emend. (, Parabasalia, , Eopharyngia) and Loukozoa emend. (Jakobea, Malawimonas): their evolutionary affinities and new higher taxa. Int. J. Syst. Evol. Microbiol. 53, 1741-1758. Cavalier-Smith, T., 2003b. Protist phylogeny and the high-level classification of Protozoa. Eur. J. Protistol. 39, 338-348. Cavalier-Smith, T., 2007. Evolution and relationships of algae: major branches of the tree of life. In Unravelling the algae - the past, present, and future of algal systematics, eds J. Brodie and J. Lewis. CRC, Boca Raton. Pp. 21-55. Cavalier-Smith, T., 2010a. Kingdoms Protozoa and and the eozoan root of eukaryotes. Biol. Lett. 6, 342-345. Cavalier-Smith, T., 2010b. Deep phylogeny, ancestral groups, and the four ages of life. Phil. Trans. Roy. Soc. B 365, 111-132.

Cavalier-Smith, T., 2013a. Early evolution of eukaryote feeding modes, cell structural diversity, and classification of the protozoan phyla Loukozoa, Sulcozoa, and Choanozoa. Europ. J. Protistol. 49, 115-178. Cavalier-Smith, T., 2013b. Symbiogenesis: mechanisms, evolutionary consequences, and systematic implications. Ann. Rev. Ecol. Evol. System. 44, 145-172. Cavalier-Smith, T., 2014a. Gregarine site-heterogeneous 18S rDNA trees, revision of gregarine higher classification, and the evolutionary diversification of Sporozoa. Eur. J. Protistol. 50, 472-495. Cavalier-Smith, T., 2014b. The neomuran revolution and phagotrophic origin of eukaryotes and cilia in the light of intracellular coevolution and a revised tree of life. in: Keeling, P.J., Koonin, E.V. (Eds.), The Origin and Evolution of Eukaryotes. Cold Spring Harbor Perspectives Biol. doi: 10.1101/cshperspect.a016006., Cold Spring Harbor. Cavalier-Smith, T. (2015). Mixed heterolobosean and novel gregarine lineage genes from culture ATCC 50646: Long-branch artefacts, not lateral gene transfer, distort -tubulin phylogeny. Europ. J. Protistol. 51, 121-137. Cavalier-Smith, T., Karpov, S.A., 2012. Paracercomonas kinetid ultrastructure, origins of the body plan of , and cytoskeleton evolution in Cercozoa. Protist 163, 47-75. Cavalier-Smith, T., Chao, E.E., Snell, E.A., Berney, C., Fiore-Donno, A.M., Lewis, R., 2014. Multigene eukaryote phylogeny reveals the likely protozoan ancestors of opisthokonts (animals, fungi, choanozoans) and Amoebozoa. Mol. Phylog. Evol. 81, 71-85. Cavalier-Smith, T., Chao, E.E., Lewis, R., 2015a. Multiple origins of from flagellate ancestors: New cryptist subphylum Corbihelia, superclass Corbistoma, and monophyly of , Cryptista, and Chromista. Mol. Phylogenet. Evol. 93, 331-362. Cavalier-Smith, T., Fiore-Donno, A.M., Chao, E., Kudryavtsev, A., Berney, C., Snell, E.A., Lewis, R., 2015b. Multigene phylogeny resolves deep branching of Amoebozoa. Mol. Phylogenet. Evol. 83, 293- 304. Cavalier-Smith, T., Chao, E.E., Vickerman, K., 2016a. New phagotrophic euglenoid species, (new genus Decastava; Scytomonas saepesedens; Entosiphon oblongum), Hsp90 introns, and putative euglenoid Hsp90 pre-mRNA insertional editing. Europ. J. Protistol. In press. Cavalier-Smith, T., Chao, E. E., Lewis, R., 2016b. 187-gene phylogeny of protozoan phylum Amoebozoa reveals a new class (Cutosea) of deep-branching, ultrastructurally unique, enveloped marine , and clarifies evolution. Mol. Phylogen. Evol. 99, 275-296. Chan, Y.F., Chiang, K.P., Chang, J., Moestrup, O., Chung, C.C., 2015. Strains of the morphospecies Ploeotia costata (Euglenozoa) isolated from the Western North Pacific (Taiwan) reveal substantial genetic differences. J. Eukaryot. Microbiol. 62, 318-326.

Chan, Y.F., Moestrup, O., Chang, J., 2013. On Keelungia pulex nov. gen. et nov. sp., a heterotrophic euglenoid flagellate that lacks pellicular plates (Euglenophyceae, Euglenida). Eur. J. Protistol. 49, 15-31. Dentler, W., 2005. Intraflagellar transport (IFT) during assembly and disassembly of Chlamydomonas flagella. J. Cell Biol. 170, 649-659. Derelle, R., Torruella, G., Klimes, V., Brinkmann, H., Kim, E., Vlcek, C., Lang, B.F., Elias, M., 2015. Bacterial proteins pinpoint a single eukaryotic root. Proc. Natl Acad. Sci. USA 112, E693-699. Deschamps, P., Lara, E., Marande, W., López-García, P., Ekelund, F., Moreira, D., 2011. Phylogenomic analysis of kinetoplastids supports that trypanosomatids arose from within bodonids. Mol. Biol. Evol. 28, 53-58. Dobáková, E., Flegontov, P., Skalický, T., Lukeš, J., 2015. Unexpectedly streamlined mitochondrial genome of the euglenozoan Euglena gracilis. Genome Biol. Evol. 7, 3358-3367. Dujardin, F., 1841. Histoire naturelle des zoophytes infusoires. Roret, Paris. Ekebom, J., Patterson, D.J., Vørs, N., 1995/6. Heterotrophic flagellates from coral reef sediments (Great Barrier Reef, Australia). Arch. Protistenk. 146, 251-272. Elbrächter, M., Schnepf, E., Balzer, I., 1996. Hemistasia phaeocysticola (Scherffel) comb. nov., redescription of a free-living, marine, phagotrophic kinetoplastid flagellate. Arch. Protistenk. 147, 125- 136. Farmer, M.A., Triemer, R.E., 1988. A redescription of the genus Ploeotia Duj. (Euglenophyceae). Taxon 37, 319-325. Farmer, M.A., Triemer, R.E., 1994. An ultrastructural study of Lentomonas applanatum (Preisig) n. g. (Euglenida). J. Euk. Microbiol. 41, 112-119. Fenchel, T., Bernard, C., Esteban, G., Finlay, B. J., Hansen, P. J., Iversen, N., 1995. Microbial diversity and activity in a Danish fjord with anoxic deep water. Ophelia 43, 45-100. Fernández-Galiano, D., 1990. Las nuevas clasificaciones de los organismos eucarióticos unicelulares protistología versus protozoología. Rev. R. Soc. Esp. Hist. Nat. (Secc. Biol.) 85, 107-125. Flegontov, P., Votýpka, J., Skalický, T., Logacheva, M.D., Penin, A.A., Tanifuji, G., Onodera, N.T., Kondrashov, A.S., Volf, P., Archibald, J.M., Lukeš, J., 2013. Paratrypanosoma is a novel early- branching trypanosomatid. Curr. Biol. 23, 1787-1793. Fritsch, F.E., 1948. The structure and reproduction of the algae. Vol. 1. Cambridge University Press, Cambridge. Frolov, A.O., Karpov, S.A., 1995. Comparative morphology of kinetoplastids. Tsitologiia 37, 1072- 1096. Frolov, A.O., Karpov, S.A., Mylnikov, A.P., 2001. The ultrastructure of Procryptobia sorokini (Zhukov) comb. nov. and rootlet homology in kinetoplastids. Protistology 2, 85-95.

Gibbs, S.P., 1981. The of some algal groups may have evolved from symbiotic green algae. Ann. N. Y. Acad. Sci. 361, 193-208. Griessmann, K., 1913. Über marine Flagellaten. Archiv Protistenk 32, 1-78. Gualdrón-López, M., Brennand, A., Hannaert, V., Quiñones, W., Cáceres, A.J., Bringaud, F., Concepción, J.L., Michels, P.A., 2012. When, how and why glycolysis became compartmentalised in the Kinetoplastea. A new look at an ancient organelle. Int. J. Parasitol. 42, 1-20. Hausmann, K., Hülsmann, N., 1996. Protozoology. Georg Thieme, Stuttgart. Hirose, E., Nozawa, A., Kumagai, A., Kitamura, S., 2012. Azumiobodo hoyamushi gen. nov. et sp. nov. (Euglenozoa, Kinetoplastea, Neobodonida): a pathogenic kinetoplastid causing the soft tunic syndrome in ascidian aquaculture. Dis. Aquat. Organ. 97, 227-235. Hollande, A., 1942. Étude cytologique et biologique de quelques flagellés libres. Arch. Zool.Exp. Gén. 83, 1-268. Hollande, A., 1952. Classe des Eugléniens (Euglenoidina Bütschli, 1884), in: Grassé, P.-P. (Ed.), Traité de Zoologie. Masson, Paris, pp. 238-284. Honigberg, B.M., Balamuth, W., Bovee, E. C., Corliss, J. O., Gojdics, M., Hall, R. P., Kudo, N. D., Levine, N. D., Loeblich Jr., A. R., Weiser, J., & Wenrich, D. H., 1964. A revised classification of phylum Protozoa. J. Protozool. 11, 7-20. Howe, A.T., Bass, D., Scoble, J.M., Lewis, R., Vickerman, K., Arndt, H., Cavalier-Smith, T., 2011. Novel cultured protists identify deep-branching environmental DNA clades of Cercozoa: new genera Tremula, Micrometopion, Minimassisteria, Nudifila, Peregrinia. Protist 162, 332- 372. Huber-Pestalozzi, G., 1955. Das Phytoplankton des Süsswassers; Sytematic und Biologie. Euglenophyceen. Teil 4. E. Schweitzerbart'sche Verlagsbuchhandlung, Stuttgart. Joyon, L., Lom, J., Etude cytologique, systématique et pathologique d‟Ichtyobodo necator (Henneguy, 1883) Pinto, 1928 (Zooflagelle). J. Protozool. 16, 703-719. Karpov, S.A.E., 2000. Protista: Handbook on zoology. Nauka, St Petersburg. Kim, J.I., Shin, W., Triemer, R.E., 2010. Multigene analyses of photosynthetic euglenoids and new family, Phacaceae (Euglenales). J. Phycol. 46, 1278-1287. Kivic, P.A., Walne, P.L., 1984. An evaluation of a possible phylogenetic relationship between the Euglenophyta and Kinetoplastida. Origins Life 13, 269-288. Klebs, G., 1883. Über die Organisation einiger Flagellaten-Gruppen und ihre Beziehungen zu Algen und Infusorien. Unters. Bot. Inst. Tübingen 1, 233-262. Klebs, G., 1892. Flagellatenstudien I and II. Zeit. wiss. Zool. 55, 265-445. Kudo, R.R., 1966. Protozoology 5th Ed. Thomas, Springfield. Lackey, J.B., 1960. Calkinsia aureus gen. et sp. nov., a new marine euglenid. Trans. Am. Microsc. Soc.

79, 105-107. Lankester, E.R., 1885. Protozoa. In: Baynes, T.S. (Ed.), The Encyclopedia Britannica, 9th ed. J. M. Stoddard Co. Ltd, Philadelphia, pp. 831–865. Larsen, J., Patterson, D.J., 1990. Some flagellates (Protista) from tropical marine sediments. J. Nat. Hist. 24, 801-937. Lasek-Nesselquist, E., Gogarten, J.P., 2013. The effects of model choice and mitigating bias on the ribosomal tree of life. Mol. Phylogenet. Evol. 69, 17-38. Lax, G., Simpson, A.G.B., 2013. Combining molecular data with classical morphology for uncultured phagotrophic euglenids (Excavata): a single-cell approach. J. Eukaryot. Microbiol. 60, 615-625. Leander, B.S., 2004. Did trypanosomatid parasites have photosynthetic ancestors? Trends Microbiol. 12, 251-258. Lee, W.J., 2002. Redescription of the rare heterotrophic flagellate (Protista) - Phyllomitus undulans Stein, 1878, and erection of a new genus - Pseudophyllomitus gen. n. Acta Protozool. 41, 375-381. Lee, W.J., Simpson, A.G.B., 2014a. Morphological and molecular characterisation of Notosolenus urceolatus Larsen and Patterson 1990, a member of an understudied deep- branching euglenid group (petalomonads). J. Eukaryot. Microbiol. 61, 463-479. Lee, W.J., Simpson, A.G.B., 2014b. Ultrastructure and molecular phylogenetic position of Neometanema parovale sp. nov. (Neometanema gen. nov.), a marine phagotrophic euglenid with skidding motility. Protist 165, 452-472. Leedale, G., 1966. Euglena: a new look with the electron microscope. Adv. Sci. 23, 22-37. Leedale, G., 1967. Euglenoid Flagellates. Prentice-Hall, Englewood Cliffs, N. J. Leedale, G., 2002 dated 2000. Class Euglenoidea Bütschli, 1884, in: Lee, J.J., Leedale, G., Bradbury, P. (Eds.), The Illustrated Guide to the Protozoa. Society of Protozoologists, Lawrence, Kansas, pp. 1136- 1157. Lemmermann, E., 1913. Flagellatae 2. Gustav Fischer, Jena. Levine, N.D., Corliss, J.O., Cox, F.E., Deroux, G., Grain, J., Honigberg, B.M., Leedale, G.F., Loeblich, A.R., 3rd, Lom, J., Lynn, D., Merinfeld, E.G., Page, F.C., Poljansky, G., Sprague, V., Vávra, J., Wallace, F.G., 1980. A newly revised classification of the protozoa. J. Protozool. 27, 37-58. Linton, E.W., Karnkowska-Ishikawa, A., Kim, J.I., Shin, W., Bennett, M.S., Kwiatowski, J., Zakrys, B., Triemer, R.E., 2010. Reconstructing euglenoid evolutionary relationships using three genes: nuclear SSU and LSU, and chloroplast SSU rDNA sequences and the description of Euglenaria gen. nov. (Euglenophyta). Protist 161, 603-619. Linton, E.W., Triemer, R.E., 1999. Reconstruction of the feeding apparatus in Ploeotia costata

(Euglenophyta) and its relationsip to other euglenoid feeding apparatuses. J. Phycol. 35, 313-324. Linton, E.W., Triemer, R.E., 2001. Reconstruction of the flagellar apparatus in Ploeotia costata (Euglenozoa) and its relationship to other euglenoid flagellar apparatuses. J. Eukaryot. Microbiol. 48, 88-94. Makiuchi, T., Annoura, T., Hashimoto, M., Hashimoto, T., Aoki, T., Nara, T., 2011. Compartmentalization of a glycolytic enzyme in Diplonema, a non-kinetoplastid euglenozoan. Protist 162, 482-489. Marande, W., Lukeš, J., Burger, G., 2005. Unique mitochondrial genome structure in diplonemids, the sister group of kinetoplastids. Eukaryot. Cell 4, 1137-1146. Marin, B., Palm, A., Klingberg, M., Melkonian, M., 2003. Phylogeny and taxonomic revision of plastid-containing euglenophytes based on SSU rDNA sequence comparisons and synapomorphic signatures in the SSU rRNA secondary structure. Protist 154, 99-145. Mayr, E., Bock, W.J., 2002. Classifications and other ordering systems. J. Syst. Evol. Res. 169-194. Mignot, J.-P., 1964. Observations complémentaires sur la structure des flagelles d'Entosiphon sulcatum (Duj.) Stein, flagellé Euglénien. Comptes R. Séances Acad. Sci. 258, 3360-3363. Mignot, J.-P., 1967. Affinités des euglénamonadines et des chloromonadines: remarques sur la sytématique des Euglénida. Protistologica 3, 25-60. Mignot, J.-P., Brugerolle, G., Bricheux, G., 1987. Intercalary strip development and dividing cell morphogenesis in the euglenoid Cyclidiopsis acus. Protoplasma 139, 51-65. Montegut-Felkner, A. E., Triemer, R. E. 1994. Phylogeny of Diplonema ambulator (Larsen and Patterson): 1. Homologies of the flagellar apparatus. Eur. J. Protistol., 30:227–237. Moreira, D., López-García, P., Vickerman, K., 2004. An updated view of kinetoplastid phylogeny using environmental sequences and a closer outgroup: proposal for a new classification of the class Kinetoplastea. Int. J. Syst. Evol. Microbiol. 54, 1861-1875. Mylnikov, A.P., 1986. Ultrastructure of a colourless flagellate, Phyllomitus apiculatus Skuja 1948 (Kinetoplastida). Arch. Protistenk. 182, 1-10. Mylnikov, A.P., Mylnikova, Z.M., Tsvetkov, A.H., Elizarova, V.A., 1998. Fine structure of the predatory flagellate Phyllomitus amylophagus (in Russian). Biol. Vnutr. Vod 2, 21-27. Pascher, A., 1931. Systematische Übersicht über die mit Flagellaten in Zusammenhang stehenden Algenreihen und versuch einen Einreihung dieser Algenstämme in die Stämme des Pflanzenreiches. Biol. Zentralbl. 48, 317-332. Patterson, D.J., Simpson, A.G.B., 1996. Heterotrophic flagellates from coastal marine and hypersaline sediments in Western Australia. Europ. J. Protistol. 32, 423-448. Perez, E., Lapaille, M., Degand, H., Cilibrasi, L., Villavicencio-Queijeiro, A., Morsomme, P., Gonzalez-Halphen, D., Field, M.C., Remacle, C., Baurain, D., Cardol, P., 2014. The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae. 19 Pt B, 338-349. Pettigrew, G.W., Leaver, J.L., Meyer, T.E., Ryle, A.P., 1975. Purification, properties, and sequences of atypical cytochrome c from two Protozoa, Euglena gracilis and oncopelti. Biochem. J. 147, 291-302. Preisig, H., 1979. Zwei neue Vertreter der farblosen Euglenophyta. Schweiz. Z. Hydrol. 41, 155-160. Pringsheim, E. G., 1948. Taxonomic problems in the Euglenineae. Biol. Rev. 23, 46-61. Raymann, K., Brochier-Armanet, C., Gribaldo, S., 2015. The two- tree of life is linked to a new root for the . Proc. Natl Acad. Sci. USA. 112, 6670-6675. doi: 10.1073/pnas.1420858112. Ritter von Stein, F., 1878. Der Organismus der Infusionsthiere. III. Der Organismus der Flagellaten I. Engelmann, Leipzig. Roy, J., Faktorová, D., Lukeš, J., Burger, G., 2007. Unusual mitochondrial genome structures throughout the Euglenozoa. Protist 158, 385-396. Ruggiero, M.A., Gordon, D.P., Orrell, T.M., Bailly, N.B., Bourgoin, T., Brusca, T., Cavalier-Smith , T., Guiry, M., Kirk, P.M., 2015. A higher level classification of all living organisms. PLOSONE 10, e0119248. DOI:10.1371/journal.pone.0119248. Saville Kent, W., 1880-1882. A manual of the Infusoria. Bogue, London. Scherfell, A., 1900. Phaeocystis globosa nov. spec. nebst einigen Betrachtungen über die Phylogenie niederer, inbesondere brauner Organismen. Wiss. Meeresunters., Abt. Helgoland 4, 1-29. Schoenichen, W. 1925. In Eyferth, B. and Schoenichen, W. Einfachste Lebensformen des Tier- und Pflanzenreiches. 5. Aufl. Band I. Spaltpflanzen, Geissellinge, Algen, Pilze. pp. vii + 519. Berlin: Lichterfelde. Scholey, J.M., 2013a. Cilium assembly: delivery of tubulin by kinesin-2-powered trains. Curr. Biol. 23, R956-959. Scholey, J.M., 2013b. Kinesin-2: a family of heterotrimeric and homodimeric motors with diverse intracellular transport functions. Ann. Rev. Cell Devel. Biol. 29, 443-469. Schroeckh, S., Lee, W. J. & Patterson, D. J. 2003. Free-living heterotrophic euglenids from freshwater sites in mainland Australia. Hydrobiologia, 493,131–166. Schwartz, R.M., Dayhoff, M.O., 1978. Origins of prokaryotes, eukaryotes, mitochondria, and chloroplasts. Science 199, 395-403. Senn, G., 1900. Eugleninae. In Engler, A., Prantl, K. eds Die natürlichen Pflanzenfamilien. 1. Engelmann, Leipzig, pp. 173–185. Senn, G., 1911. Oxyrrhis, Nephroselmis und einige Euflagellaten, nebst Bemerkungen über deren System. Zschr. wiss. Zool. 97, 605-672.

Shih, S.M., Engel, B.D., Kocabas, F., Bilyard, T., Gennerich, A., Marshall, W.F., Yildiz, A., 2013. Intraflagellar transport drives flagellar surface motility. eLife 2, e00744. Shin, W., Brosnan, S., Triemer, R.E., 2002. Are cytoplasmic pockets (MTR/pocket) present in all photosynthetic euglenoid genera. J. Phycol. 38, 790-799. Shiratori, T., Yokoyama, A., Ishida, K., 2014. Phylogeny, ultrastructure, and flagellar apparatus of a new marimonad flagellate Abollifer globosa sp. nov. (Imbricatea, Cercozoa). Protist 165, 808-824. Simpson, A.G.B., 1997. The identity and composition of the Euglenozoa. Arch. Protistenk. 148, 318-328. Simpson, A.G.B., Inagaki, Y., Roger, A.J., 2006. Comprehensive multigene phylogenies of excavate protists reveal the evolutionary positions of "primitive" eukaryotes. Mol. Biol. Evol. 23, 615-625. Simpson, A.G., Lukeš, J., Roger, A.J., 2002. The evolutionary history of kinetoplastids and their kinetoplasts. Mol. Biol. Evol. 19, 2071-2083. Simpson, A.G., Roger, A.J., 2004. Protein phylogenies robustly resolve the deep-level relationships within Euglenozoa. Mol. Phylogenet. Evol. 30, 201-212. Simpson, A.G.B., van den Hoff, J., Bernard, C., Burton, H.R., Patterson, D.J., 1996/7. The ultrastructure and systematic position of the euglenozoon Postgaardi mariagerensis, Fenchel et al. Arch. Protistenk. 147, 213-225. Skuja, H., 1934 dated 1932. Beitrag zur Algenflora Lettlands I. Acta Hort. Bot. Univ. Latt. 7, 25-85. Smirnov, A.V., Chao, E., Nassonova, E.S., Cavalier-Smith, T., 2011. A revised classification of naked lobose amoebae (Amoebozoa: Lobosa). Protist 162, 545-570. Smith, G.M., 1933. The freshwater algae of the United States. McGraw-Hill. New York and London. Stoeck, T., Schwarz, M.V., Boenigk, J., Schweikert, M., von der Heyden, S., Behnke, A., 2005. Cellular identity of an 18S rRNA gene sequence clade within the class Kinetoplastea: the novel genus Actuariola gen. nov. (Neobodonida) with description of the type species Actuariola framvarensis sp. nov. Int. J. Syst. Evol. Microbiol. 55, 2623-2635. Taylor, F.J.R., 1976. Flagellate phylogeny: a study in conflicts. J. Protozool. 23, 28-40. Triemer, R.E., 1986. Light and electron microscopic description of a colourless euglenoid, Serpenomomas costata n. g., n. sp. J. Protozool. 33, 412-415. Triemer, R.E., Farmer, M.A., 1991a. An ultrastructural comparison of the mitotic apparatus, feeding apparatus, flagellar apparatus and cytoskeleton in euglenoids and kinetoplastids. Protoplasma 164, 91- 104.

Triemer, R.E., Farmer, M.A., 1991b. The ultrastructural organization of heterotrophic euglenids and its evolutionary implications, in: Patterson, D.J., & J. Larsen (Ed.), The biology of free- living heterotrophic flagellates. Clarendon Press, Oxford, pp. 185-204. Triemer, R.E., Farmer, M. A., 2007. A decade of euglenoid molecular systematics. In Unravelling the algae - the past, present, and future of algal systematics, eds J. Brodie and J. Lewis. CRC, Boca Raton. Pp. 315-330. Triemer, R.E., Fritz, L., 1988. Ultrastructural features of mitosis in Ploeotia costata (Heteronematales, Euglenophyta). J. Phycol. 24, 514-519. Vasileva, I.I., 1987. Yakut euglenoid and xanthophyte algae. Nauka, Leningrad. (in Russian). von der Heyden, S., Chao, E.E., Vickerman, K., Cavalier-Smith, T., 2004. Ribosomal RNA phylogeny of bodonid and diplonemid flagellates and the evolution of Euglenozoa. J. Euk. Microbiol. 51, 402-416. von der Heyden, S., Cavalier-Smith, T., 2005. Culturing and environmental DNA sequencing uncover hidden kinetoplastid biodiversity and a major marine clade within ancestrally freshwater Neobodo designis. Int. J. Syst. Evol. Microbiol. 55, 2605-2612. Walne, P.L., Kivic, P.A., 1990. Phylum Euglenida, in: Margulis, L., Corliss, J.O., Melkonian, M., Chapman, D.J. (Eds.), Handbook of Protoctista. Jones & Bartlett, Boston, pp. 270-287. Walton, L.B., 1915. A review of described species of the order Euglenoidina Bloch, Class Flagellata (Protozoa) with particular reference to those found in the city water supplies and in other localities in Ohio. Ohio State Univerity Bull. 19, 341-449. West, G. S., 1904. A Treatise on the British Freshwater Algae. The University Press, Cambridge. West, G. S., Fritsch, F.E., 1932. A Treatise on the British Freshwater Algae. Revised Ed. The University Press, Cambridge. Yabuki, A., Tame, A., 2015. Phylogeny and reclassification of Hemistasia phaeocysticola (Scherffel) Elbrächter & Schnepf, 1996. J. Eukaryot. Microbiol. 62, 426-429. Yamaguchi, A., Yubuki, N., Leander, B.S., 2012. Morphostasis in a novel eukaryote illuminates the evolutionary transition from phagotrophy to phototrophy: description of Rapaza viridis n. gen. et sp. (Euglenozoa, Euglenida). BMC Evol. Biol. 12, 29. Yubuki, N., Edgcomb, V.P., Bernhard, J.M., Leander, B.S., 2009. Ultrastructure and molecular phylogeny of Calkinsia aureus: cellular identity of a novel clade of deep-sea euglenozoans with epibiotic bacteria. BMC Microbiol. 9, 16. Yubuki, N., Simpson, A.G., Leander, B.S., 2013. Reconstruction of the feeding apparatus in Postgaardi mariagerensis provides evidence for character evolution within the Symbiontida (Euglenozoa). Eur. J. Protistol. 49, 32-39.

Figure 1. PhyloBayes CAT-GTR-GAMMA tree for 18S rDNA of 282 eukaryotes (224 Euglenozoa) and 1577 nucleotide positions. Both chains (each run for nearly half a million genrations) converged to the same topology (maxdiff 0.14; 163,998 trees summed, excluding first 1604 as burnin). Newly sequenced taxa are in bold. Support values for bipartitions are posterior probabilities (left) and bootstrap percentages for 1000 fast bootstraps for a RAxML GTR gamma analysis for the same alignment (right). Black blobs mean maximal (1.0, 100%) support by both methods, for example that between Euglenozoa and all other eukaryotes. Taxa for some outgroups whose internal phylogeny is irrelevant to this paper are collapsed to enable fitting onto one page; the number of species for each is beside its name; their names and internal topology are on an uncollapsed 323-taxon tree that included Entosiphon and Percolozoa (Fig. S2). The Entosiphon branch is from the equivalent CAT-GTR-GAMMA tree with 287 taxa; its ultra-long stem has been halved in length, the arrow marked E and dashed line showing where it joined that tree, the rest of whose topology is identical to this except for the position of Dinema sulcatum that grouped with other anisonemids (see text); when Percolozoa are included in the outgroup Entosiphon moves down one node to be sister to Dipilida and Teloprocta becomes sister of Anisonemia (Fig. S2). Tree-rooting follows Cavalier-Smith (2010a, 2013a, 2014b). Clade names follow the taxonomy of Table 1.

Figure 2. Schematic relationships amongst the major euglenozoan taxa as shown by 18S rDNA (Figs 1, S1, S2) and Hsp90 trees (Cavalier-Smith et al. 2016a). Eutreptiida, Bodonida, Rigimonada, and Peranemea are known to be paraphyletic and ancestral to the groups indicated. All other named taxa are probably clades, though a minority of my trees (e.g. Fig. 1) raise the possibility that Anisonemida might be paraphyletic ancestors of Natomonadida because of deeper branching of Dinema scaphurum (most, as Cavalier-Smith et al. (2016a) explained, show Anisonemida as a clade, e.g. Fig. S1), and only one sequence each is known for Ploeotiida and Acroglissida (Teloprocta); more are needed to test this. There is also uncertainty whether Acroglissida are sister to Euglenophyceae as shown (e.g. Fig. 1) or to Anisonemia (dashed line and Fig. S2).

Table 1. Classification of phylum Euglenozoa and its 8 classes, 18 orders, and 31 families Subphylum 1. Euglenoida Bütschli, 1884 (as Euglenoidina) stat. n. Cavalier-Smith, 1993 (aerobes; pellicular strips) Infraphylum 1. Entosiphona infraphyl. n. (three feeding apparatus support rods) Class Entosiphonea cl. n. (posterior ciliary gliders)

Order Entosiphonida ord. n. Family Entosiphonidae fam. n. (Entosiphon) Infraphylum 2. Dipilida infraphyl. n. (two feeding apparatus support rods) Superclass 1. Rigimonada* supercl. n. (rigid longitudinal pellicle strips) Class 1. Stavomonadea cl. n. (simple strip joints, not below prominent ridges) Subclass 1. Homostavia subcl. n. (uniform pellicle strips) Order 1. Decastavida Cavalier-Smith in Cavalier-Smith et al., 2016a (posterior gliders) Family 1. Decastavidae Cavalier-Smith in Cavalier-Smith et al., 2016a (Decastava) Family 2. Keelungiidae fam. n. (Keelungia) Order 2. Petalomonadida Cavalier-Smith, 1993 (anterior gliders) Family 1. Scytomonadidae Ritter von Stein, 1878 (syn. Petalomonadidae Bütschli, 1884) (Atraktomonas, Biundula, Calycimonas, Dolium, Dylakosoma, Notosolenus, Pentamonas, Petalomonas, Scytomonas, Tropidoscyphus) Family 2. Sphenomonadidae Saville Kent, 1880 (Sphenomonas) Subclass 2. Heterostavia subcl. n. (heteromorphic strips) Order Heterostavida ord. n. (posterior gliders) Family Serpenomonadidae fam. n. (Serpenomonas) Class 2. Ploeotarea cl. n. (prominent dorsal pellicle ridges and 10 microgrooves) Order Ploeotiida Cavalier-Smith, 1993 (posterior gliders) Family 1. Ploeotiidae fam. n. (Ploeotia) Family 2. Lentomonadidae fam. n. (Lentomonas) Superclass 2. Spirocuta supercl. n. (spiral pellicle strips; often squirm) Class 1. Peranemea Cavalier-Smith, 1993 em. (heterotrophs) Subclass 1. Peranemia subcl. n. (protrusible rod apparatus) Order Peranemida Cavalier-Smith, 1993 (anterior gliders) Family Peranemidae Bütschli, 1884 (Peranema, Peranemopsis, Jenningsia, Urceolus) Subclass 2. Anisonemia subcl. n. (non-protrusible rod apparatus) Order 1. Anisonemida ord. n. (posterior gliders) Family Anisonemidae Saville Kent, 1880 em. (Anisonema, Dinema*, Heteronema) Order 2. Natomonadida ord. n. (swimmers)

Suborder 1. Metanemina subord. n. (phagotrophs) Family Neometanemidae fam. n. (Neometanema) Suborder 2. Rhabdomonadina Leedale, 1967 em. Cavalier-Smith, 1993 (osmotrophs: originally excluded Astasia and Distigma) Family 1. Astasiidae Saville Kent, 1884 (as Astasiadae; incl. Menoidiidae Hollande, 1942) (Astasia, Rhabdomonas, Gyropaigne, Menoidium, Parmidium) Family 2. Distigmidae Hollande, 1942 (some violently plastic others rigid) (Distigma) Subclass 3. Acroglissia subcl. n. (anterior gliders) Order 1. Acroglissida ord. n. Family Teloproctidae Cavalier-Smith in Cavalier-Smith et al., 2016a (Teloprocta) Class 2. Euglenophyceae Schoenichen in Eyfurth and Shoenichen, 1925 orth. em. Smith, 1933** (ancestrally and largely phototrophs) Subclass 1. Rapazia subcl. n. (phagophototrophs) Order Rapazida ord. n. (16 pellicular strips) Family Rapazidae fam. n. (Rapaza) Subclass 2. Euglenophycidae Busse and Preisfeld, 2003 (non-phagotrophs) Order 1. Eutreptiida* Leedale, 1967 (=Eutreptiales; 48 pellicle strips) Family Eutreptiidae* Hollande, 1942 (Eutreptia*, Eutreptiella, Tetreutreptia) Order 2. Euglenida Ritter von Stein, 1878 stat. n. Calkins, 1926 (=Euglenales Leedale, 1967; 40 pellicular strips, two posterior whorls) *** Family 1. Euglenidae Dujardin, 1841 em. Kim et al., 2010 1. Eugleninae Hollande, 1952 em. (Euglena, Euglenaria, Khawkinea, Monomorphina, ) Subfamily 2. Colaciinae Smith, 1933 (as family) (Colacium, Strombomonas, ) Family 2. Phacidae Kim et al., 2010 (=Phacaceae) (, , Discoplastis) Family 3. Euglenamorphidae Hollande, 1952 (as subfamily) stat. n. (Euglenamorpha, Hegneria) Subphylum 2. Postgaardia subphyl. n. (anaerobes; epicellular bacteria; strips, glycosomes absent)

Class Postgaardea Cavalier-Smith, 1998 (synonym Symbiontida Yubuki et al., 2009; no rank) Order 1. Postgaardida Cavalier-Smith, 2003 Family 1. Postgaardidae fam. n. (Postgaardi) Family 2. Calkinsiidae fam. n. (Calkinsia) Order 2. Bihospitida ord. n. Family Bihospitidae fam. n. (Bihospites) Subphylum 3. Glycomonada subphyl. n. (heterotrophs with glycosomes, ancestrally phagotrophs) Class 1. Diplonemea Cavalier-Smith, 1993 Order Diplonemida Cavalier-Smith, 1993 Family 1. Diplonemidae Cavalier-Smith, 1993 (Diplonema, Rhizopus) Family 2. Hemistasiidae fam. n. (Hemistasia) Class 2. Kinetoplastea Honigberg em. Vickerman, 1976 stat. n. Margulis, 1974 Subclass 1. Prokinetoplastina Vickerman in Moreira et al., 2004 Order 1. Prokinetoplastida Vickerman in Moreira et al., 2004 Family Ichthyobodonidae Isaksen et al., 2007 (Ichthyobodo, ) Subclass 2. Metakinetoplastina Vickerman in Moreira et al., 2004 Order 1. Bodonida* Hollande, 1952 em. Vickerman, 1976, Krylov et al. 1980 Suborder 1. Neobodonina Vickerman in Moreira et al., 2004 stat. n. Family 1. Neobodonidae* fam. n. (Neobodo*, Rhynchobodo, Actuariola, Azumiobodo, Cruzella, Cryptaulaxoides, Klosteria) Family 2. Rhynchomonadidae fam. n. (Rhynchomonas, Dimastigella) Suborder 2. Parabodonidina Vickerman in Moreira et al., 2004 stat. n. Family 1. Parabodonidae fam. n. (Parabodo) Family 2. Cryptobiidae* Vickerman, 1976 (Cryptobia*, Procryptobia, Trypanoplasma, Cephalothamnion) Suborder 3. Eubodonidina Vickerman in Moreira et al., 2004 stat. n. Family Bodonidae Bütschli, 1883 em. (Bodo) Order 2. Trypanosomatida Saville Kent, 1880 stat. n. Hollande, 1952 Family Trypanosomatidae Doflein, 1901 (e.g. , Crithidia, , , Paratrypanosoma, , Sergeia, , )

* Probably paraphyletic

** I avoided the much older potential name Euglenea Bütschli, 1884 for Euglenophyceae, used contradictorily as a synonym for all Euglenophycidae by Busse and Preisfeld (2003) and by Adl et al. (2012) for Euglenidae only, as it was also published as a beetle genus in 1896. There is no good reason to regard any Euglenozoa except class Euglenophyceae as algae, which are best defined as 'oxygenic photosynthesisers other than embryophyte land plants' (Cavalier-Smith 2007); it is simplest to treat all Euglenozoa including Euglenophyceae uniformly under the Zoological Code of Nomenclature, under which the names Dinema, Entosiphon, and Peranema are valid; thus younger replacement names Dinematomonas, Entosiphonomonas and Pseudoperanema, are entirely unnecessary, contrary to some statements, and their use is nomenclaturally destabilizing. If botanists wish for historical reasons to treat photosynthetic euglenoids under the International Code of Nomenclature for algae, fungi and plants, that is not unreasonable provided that this policy is strictly limited to Euglenophyceae as circumscribed here (as it includes Rapazida, broader than Euglenophycidae of Busse and Preisfeld (2003) and compositionally equivalent Euglenophyceae sensu Marin et al. 2003). The botanical division/phylum name Euglenophyta Pascher, 1931 should not be applied to Euglenozoa or Euglenoida as a whole as it is profoundly misleading for either of these ancestrally phagotrophic, non-photosynthetic, non-algal protozoan taxa. I considered using Euglenidea, already in use decades ago (e.g. Fernández-Galiano 1990), as a „zoological‟ tradition class name instead of Euglenophyceae, but settled on the latter as it should be more acceptable to both phycologists and protozoologists and thus promote future stability. *** Euglenida of Ritter von Stein (1878) was ranked as a family and referred only to Euglenidae, not all euglenoids, whose unity was first recognized by Bütschli (1884/5) under the name Euglenoidina accepted as an order by many from Blochmann (1895) through Walton (1915) to Kudo (1966) and class or higher by Hollande (1952), Leedale (1967), and numerous more recent authors, e.g. Cavalier-Smith (1993, 1998); Hausmann and Hülsmann (1996), and Karpov (2000). The traditional vernacular term euglenoids (Walton 1915) applies to all suphylum Euglenoida. Now disused early spellings (Eugleninae Lemmerman, 1913; Euglenineae) predate general use of rank-informative suffixes and are only of historical interest. A recent fashion (e.g. Adl et al. 2012) for spelling all euglenoids Euglenida (possibly related to Calkins' (1926) influence on American protozoologists and their slowness to accept a rank above order (Honigberg 1964; Levine et al. 1980), as always favoured by phycologists and most other protozoologists since 1952, or keeping that ordinal suffix even at phylum rank (Walne and Kivic 1990) causes confusion with the much narrower order Euglenida (and still more restricted family Euglenidae). The vernacular „euglenids‟ is similarly ambiguous, but might usefully be restricted in scope to refer just to order Euglenida.

Table 2. Sensitivity of Postgaardea placement to sequence sampling and substitution model ______No Percolozoa in outgroup Percolozoa in outgroup

Nucleotide positions Entosiphon Entosiphon and algorithm - (282 taxa) + (287 taxa) - (318 taxa) + (323 taxa)

______

1425 positions CAT E (0.40) E (0.43) E (0.52) E (0.40) ML G (30%) G (26%) E (45%) E (40%) 1577 positions CAT E (0.53) E (0.52) E (0.67) E (0.48) ML G (25%) E (46%) E (70%) Ento (40%) ______

Position of Postgaardea on 16 18S rDNA trees: E = sister to Euglenoida; G = sister to Glycomonada; Ento = sister to Entosiphon. The differences between E and G do not really imply a difference in topology within Euglenozoa; they simply reflect where the chosen outgroup joins the tree and therefore are a rooting rather than internal branching uncertainty; ML is clearly more sensitive to outgroup choice. Trees were run including (+) or excluding (-) Entosiphon. Support values (PP or BS %) are in brackets. ______

Table 3. Sensitivity of Teloprocta and Entosiphon placement to sequence sampling and model ______No Percolozoa in outgroup Percolozoa in outgroup

Nucleotide positions Entosiphon Entosiphon and algorithm - (282 taxa) + (287 taxa) - (318 taxa) + (323 taxa)

______

1425 positions CAT T: EP (0.66) EP (0.65) A (0.54) EP (0.39) E: PS (0.35) Sp (0.44) ML T: EP (53%)EP (53%) EP (49%) EP (48%) E: K (27%) K (32%) 1577 positions CAT T: EP (0.61) EP (0.63) A (0.66) A (0.75) E: St (0.56) D (0.35) ML T: EP (53%) R (21%) EP (49%) EP (50%) E: K (22%) P (30%) ______

Position of Teloprocta (T: shown in bold) on 16 18S rDNA trees: EP = sister to Euglenophyceae; A = sister to Anisonemia; R = sister to Rhabdomonadina.

Position of Entosiphon (E:) on 8 18S rDNA trees: PS = sister to Ploeotaria plus Spirocuta; Sp = sister to Spirocuta; D = sister to all other euglenoids (Dipilida) St = sister to Stavomonadea; K = sister to Keelungia; P = sister to Postgaardea. Trees were run including (+) or excluding (-) Entosiphon. Support values (PP or BS %) are in brackets.

0.64/- Entosiphon oblongum type strain Entosiphon oblongum TCS-2003 AY425008 X2 0.65/75 activated sludge DNA Q4C041QESU GU920505 ENTOSIPHONA 1/100 Entosiphon sulcatum CCAP 1220/1B AY061999 Entosiphon sulcatum CCAP 1220/1A AF220826 0.73/68 Decastava edaphica Decastavida 0.73/59 E Keelungia pulex Scytomonas saepesedens Arctic oxygen-depleted marine tidal sediment DNA D2P04B10 EF100248 .94/80 0.99/98 Petalomonas cantuscygni CCAP 1259/1 AF386635 0.98/66 Petalomonas cantuscygni U84731 Petalomonas cantuscygni 0.47/34 0.94 deep sea microbial mat cold seep DNA RM2-SGM31 AB505539 U /- Arctic oxygen-depleted marine tidal sediment DNA D3P06F06 EF100316 freshwater sediment DNA CH1_S2_16 AY821957 freshwater sediment DNA CH1_S2_19 AY821958 Petalomonadida G peat bog water DNA PR3_3E_63 GQ330643 Stavomonadea 0.99/100 0.6/51 Biundula (=Petalomonas) sphagnophila 3 0.99/70 freshwater sediment DNA CH1_S1_57 AY821956 L isolate U1 KC990930 Notosolenus urceolatus AF403149 Notosolenus ostium Serpenomonas aff. costata KF586333 DIPILIDA 0.98/80 E Serpenomonas aff. costata KF586332 Heterostavida E Serpenomonas costata CCAP 1265/1 Ploeotiida Ploeotarea Ploeotia cf. vitrea KC990993 Montegut-Felkner and Triemer strain CCAP 1260/1B AF386636 Peranema trichophorum Peranemida Peranemea N Dinema sulcatum AY061998 Dinema platysomum 0.99 U3 Anisonema/Dinema sp. KC990932 Anisonemia 0.99/100 0.99/100 BIPA 2001 misidentified as Peranema sp. AY048919 0.79/49 /63 G1 Anisonema sp. KC990936 O W1 Anisonema sp. KC990935 Anisonemida 0.99/100 B1 Anisonema acinus KC990937 0.93/63 Anisonema acinus AF403160 Spirocuta 0.48/- Neometanema parovale KJ690254 Metanemina I Neometanema (=Heteronema) cf. exaratum Distigma proteus var. longicauda Distigma gracilis SAG 216.80 AF386637 0.99/42 Distigma gracilis CCAP 1216/2 AY061997 0.99/99 Astasia curvata 4 D Rhabdomonadina Astasia torta Menoidium gibbum 3 Natomonadida 0.91/75 0.99/100 Rhabdomonas intermedia Parmidium circulare Parmidium scutulum 0.99/89 A .99 Rhabdomonas incurva AF403151 0.99/82 /92 Rhabdomonas intermedia AF247601 Gyropaigne costata AF110418 E Teloprocta (=Heteronema) scaphurum Rhabdomonas costata AF295021 Rapaza viridis Acroglissida Eutreptiella gymnastica AJ532400 Rapazia 0.61/53 Eutreptiella eupharyngea U Eutreptiella pomquetensis CCMP 1491 AJ532398 0.99/95 0.96/100 AJ532397 Eutreptiella braarudii Eutreptiida Eutreptiella braarudii Eutreptiella CCMP389 0.99/100 Discoplastis sp. FJ719606 G Discoplastis spathyrhyncha AJ532454 0.53/- Phacus limnophila 0.8/61 Euglenida Phacus platalea Phacus acuminatus 0.98/34 0.99 Phacus tortus L 0.99/100 Phacus ranula /73 .5/- Phacus warszewiczii Phacus pleuronectes var. minimus Phacus skujae E 0.99/63 1/98 Phacus oscillans 1/84 .94 Phacus orbicularis 0.98/25 /25 .43/- Phacus swirenkoi Phacus acuminatus var. triquetra 0.79/- Phacus segretii N . 74/- Phacus hamelii Euglenophyceae Phacus applanatus Euglenophycidae 0.99/58 .99/70 Lepocinclis salina Lepocinclis 4 1/95 Euglenaformis proxima O Monomorphina aenigmatica .99 Monomorphina pseudopyrum /46 Monomorphina rudicola Monomorphina inconspicua 0.99/81 Monomorphina ovata Z 0.99/56 Monomorphina sp. MI 73 Euglenaria sp. JQ691737 Euglena longa 0.97/95 Euglena hiemalis O Euglena gracilis 0.95/60 0.45/- Cryptoglena skujae Cryptoglena pigra 1/97 Trachelomonas zorensis Trachelomonas sp. T603 A 0.84/21 Strombomonas triquetra Strombomonas costata.98/97 0.99/100 Strombomonas ovalis 0.54/- 0.46/- Strombomonas borysthensis 0.99/100 Calkinsia aureus Colacium 70.99/100 environmental DNA 6 POSTGAARDIA marine DNA from super-sulfidic anoxic fiord water DQ310255 marine DNA clade Postgaardida 0.91/100 marine DNA from sulfidic suboxic Black Sea water HM749952 Postgaardea 0.99/100 HM004354 HM004353 Bihospites baccati Bihospitida (synonym Symbiontida) 0.99/63 Hemistasia phaeocysticola marine DNA 5 0.99/100 Hemistasidae G 0.99/100 Diplonema sp. ATCC 50224 deep ocean DNA DQ504350 L sp. ATCC 50229 AY425014 0.99/63 0.8/81 Rhynchopus euleeides ATCC 50226 0.52/83 Diplonemea Diplonema sp. ATCC 50232 Diplonemidae Y 0.42/- Diplonema sp. ATCC 50225 0.94/54 Diplonema ambulator ATCC 50223 AF30522 C 0.98/98 AF30521 Mid Atlantic Ridge sediment DNA 0.88/52 unidentified marine clade 0.82/93 Paratrypanosoma confusum Trypanosomatida O other Trypanosomatina 20 0.99/97 0.50/69 Parabodonina 8 0.99/86 Bodonida 0.99/100 .38/- Eubodonina 12 . 86 /71 + Neobodo saliens 0.21/- 0.99/100 AY998650 'Neobodo designis' SCCAP BD54 M AY998651 'Neobodo designis' SCCAP BD55 Metakinetoplastina AY490220Neobodo Auckland 0.99/100 O .99/94 DQ207578 Neobodo designis HFCC8 brackish Baltic 0.51/- AY753625 Neobodo Lake sediment Panama2 .99 Neobodo AY753610 Lake sediment San Francisco .68/85 Kinetoplastea N 0.99/100 0.51/- /99 AY753618 Neobodo marine Raglan1 .99/1 DQ207589 Neobodo saliens HFCC11 0.68/55 .99/71 AY753600 Neobodo Devon 2 marine rockpool A AY425021 Cryptaulaxoides-like Costa Rica .93/73 0.52/- Rhynchobodo 4.98/70 D 0.37/- Neobodo designis 20 .42/- 1 Dimastigella 4 Neobodonina 0.27/- Rhynchomonas nasuta 4 A 0.96/100 Perkinsiella-like AFSM3 AY163355 0.99/90 Ichthyobodo 7 Prokinetoplastina Jakobina 5 Tsukubamonas globosa Tsukubamonadea .54/30 anathema Anaeromonadea 3 Neozoa 43 0.97/- .98/84 0.41/- neokaryotes Malawimonas jakobiformis Malawimonadea 0.97/90 Andalucina 4 0.3 18 orders 8 classes 3 subphyla Rigimonada Entosphonida Entosiphonea Decastavida Petalomonadida Stavomonadea Heterostavida Ploeotiida Ploeotarea Peranemida Euglenoida Anisonemida Dipilida Peranemea Natomonadida Spirocuta Acroglissida Rapazida chloroplast Eutreptiida Euglenophyceae Euglenophycidae Euglenida Postgaardida Bihospitida Postgaardea Postgaardia Diplonemida Diplonemea Prokinetoplastida Glycomonada Bodonida Metakinetoplastina Kinetoplastea Trypanosomatida