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Protist, Vol. 161, 603–619, October 2010http://www.elsevier.de/protisPublished online date 22 April 2010

ORIGINAL PAPER

Reconstructing Euglenoid EvolutionaryRelationships using Three Genes: Nuclear SSUand LSU, and Chloroplast SSU rDNA Sequencesand the Description of Euglenaria gen. nov.(Euglenophyta)

Eric W. Lintona,1, Anna Karnkowska-Ishikawac, Jong Im Kimd, Woongghi Shind,Mathew S. Bennettb, Jan Kwiatowskic,e, Bozena Zakrysc, and Richard E. Triemerb

aCentral Michigan University, Department of Biology, Mt. Pleasant, MI 48859, USAbMichigan State University, Department of Plant Biology, East Lansing, MI 48824, USAcUniversity of Warsaw, Department of Plant Systematics and Geography, Institute of Botany, Al. Ujazdowskie 4,00-478 Warsaw, Poland

dChungnam National University, Department of Biology, 220 Gung-Dong, Yuseong-Gu Daejeon 305-764,Republic of Korea

eUniversity of California, Department of Ecology and Evolutionary Biology, 453 Steinhaus Hall, Irvine,CA 92697, USA

Submitted September 24, 2009; Accepted February 28, 2010Monitoring Editor: Michael Melkonian

Using Maximum Likelihood and Bayesian analyses of three genes, nuclear SSU (nSSU) andLSU (nLSU) rDNA, and chloroplast SSU (cpSSU) rDNA, the relationships among 82 plastid-containingstrains of euglenophytes were clarified. The resulting tree split into two major clades: cladeone contained Euglena, Trachelomonas, Strombomonas, Colacium, Monomorphina, Cryptoglenaand Euglenaria; clade two contained Lepocinclis, Phacus and Discoplastis. The majority of themembers of Euglena were contained in clade A, but seven members were outside of thisclade. Euglena limnophila grouped with, and was thus transferred to Phacus. Euglena proxima wasa single taxon at the base of clade one and is unassociated with any subclade. Five members ofEuglena grouped together within clade one and were transferred into the newly erected genusEuglenaria. The monophyly of the remaining genera was supported by Bayesian and MaximumLikelihood analyses. Combining datasets resolved the relationships among ten genera ofphotosynthetic euglenoids.& 2010 Elsevier GmbH. All rights reserved.

Key words: Bayesian inference; euglenophyta; LSU; maximum likelihood; SSU.

Introduction

Euglenoids are a group of protozoans that mostcommonly have two flagella, one or both may beemergent. Members live in salt, brackish or fresh

1Corresponding author; fax þ1 11 989 772 3462.e-mail eric.linton@cmich.edu (E.W. Linton).

& 2010 Elsevier GmbH. All rights reserved.doi:10.1016/j.protis.2010.02.002

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water environments. Many are phagotrophic orosmotrophic, however, some euglenoids are alsoconsidered algae because they are photosyn-thetic, having acquired a chloroplast via second-ary endosymbiosis of a eukaryotic green alga(Gibbs 1978; Ishida et al. 1997; Leander 2004;Martin et al. 1992; Nozaki 2005). Our research isfocused on phylogenetic (evolutionary) relation-ships among the photosynthetic euglenoids with asingle emergent flagellum, while members of theoutgroup have two emergent flagella. Because oftheir long and intricate history, a brief review ofeuglenoid morphology and taxonomy is war-ranted. An in-depth review of euglenoid classifica-tion and molecular phylogenetics can be found inTriemer and Farmer (2007).

Since the genus Euglena was first described byEhrenberg (1830a, 1830b), their classification byvarious authors has largely been based onmorphological features such as: chloroplast typeand distribution, flagellar length, shape and dis-tribution of the storage product, and cell surfacefeatures. Therefore, an understanding of eugle-noid morphological features is necessary for theirproper classification into genera.

The unique surface feature of all euglenoids isthe pellicle, a series of interlocking protein stripssubtended by microtubules that lies below theplasma membrane. If the cell is rigid or capable ofa slight bending motion, the pellicle strips can bearranged either longitudinally or helically. How-ever, all cells capable of metaboly, a peristalticwriggling motion (Triemer et al. 2006), havehelically arranged pellicle strips. Underlying thepellicle strips of some euglenoids are membrane-bounded mucus secreting bodies known asmucocysts, sensu Mignot (1966). Mucocysts,when present, can be round or spindle-shaped,thus providing a diagnostic character for manyspecies.

An exceedingly useful phylogenetic character ofall photosynthetic euglenoids is the chloroplast.The chloroplast envelope consists of three mem-branes, indicating their secondary endosymbioticorigin, while the inner photosynthetic membranes(thylakoids) occur in stacks of three and lack thegrana stacks found in land plants. The chloroplastcan be absent, reduced to a colorless plastid insome secondary osmotrophic euglenoids, orcontain chlorophyll and take on a variety ofshapes, such as discoid, lenticular, lobed, sphe-rical, ribbon-like or stellate. While the samechloroplast shape can be found in a variety ofdifferent euglenoid species, the shape for anyparticular species is specific and in some cases

specific for the whole genus. The pyrenoid, aproteinaceous area within the chloroplast contain-ing Rubisco, is associated with the formation ofthe storage products. In euglenoids this product isparamylon a beta 1,3-glucan storage compound.This structure, like the chloroplast, is oftendiagnostic of a species or an entire genus ofeuglenoids. When present, the pyrenoid may becapped by paramylon on one side (haplopyre-noids), both sides (diplopyrenoid), or lacking aparamylon cap (naked). Additional morphologicalfeatures such as cell length, shape, degree ofcompression (flatness), presence and shape of thelorica (a protective extracellular matrix that sur-rounds cell) have also been used to define speciesand genera of euglenoids.

By 1849, taxonomists had erected six of theten photosynthetic euglenoid genera analyzedin this paper. The first six genera were Euglena(Ehrenberg 1830a, 1830b, 1838), Cryptoglena(Ehrenberg 1831), Trachelomonas and Colacium(Ehrenberg 1833), Phacus (Dujardin 1841) andLepocinclis (Perty 1849). The remaining fourgenera were separated from these original generabased on more detailed examinations of morpho-logical features or on significant molecular differ-ences. Using morphological differences Mono-morphina was split off from Lepocinclis byMereschkowsky (1877), but the description ofthe genus was not recognized in the Englishliterature until recently (Marin et al. 2003). Differ-ences in the structure of the lorica were used byDeflandre (1930) to erect Strombomonas from asubgroup of Trachelomonas. The use of moleculardata, predominantly nuclear small subunit rDNA(nSSU), and modern phylogenetic methods (Max-imum Likelihood and Bayesian analyses) usheredin a new era of euglenoid systematics.

In this new era, early molecular evidencenoted that some genera (Euglena, Phacus andLepocinclis) were not monophyletic (Linton et al.1999, 2000; Milanowski et al. 2001; Mullner et al.2001) which led to several taxonomic revisions. In2003, Marin et al. resurrected Monomorphina andmoved several former Phacus and a few Lepocin-clis species into the genus thus making bothgenera monophyletic. In addition, several Euglenaspecies that were nested with taxa from Lepocin-clis were moved into this genus. Strombomonaswas then dissolved back into Trachelomonas.More recently, Triemer et al. (2006) usingincreased taxon sampling and data from multiplegenes, re-established Trachelomonas and Strom-bomonas as independent genera and erected thenew genus Discoplastis to contain two species

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formerly assigned to Euglena. Our study uses 82taxa and an expanded dataset of three genes, twonuclear (nSSU and nLSU rDNA) and one plastid(cpSSU rDNA), to resolve relationships and defineten genera, nine previously described and onenewly erected genus Euglenaria, containing fiveformer members of Euglena.

Results

Phylogenetic Analysis

Eighty-two taxa representing 12 genera (10ingroup and two outgroup) were analyzed usinga combination of three genes. Two (nSSU andnLSU) were nuclear encoded and one (cpSSU)was encoded in the chloroplast or plastid (Euglenalonga) for a total of 5161 bases used in theanalysis (nSSU: 1740, nLSU: 2110 and cpSSU:1311). Of the 246 sequences used in this study 92were previously unpublished (nSSU: 12, nLSU: 22and cpSSU: 58). Bayesian (single and partitionmodel) and Maximum Likelihood (ML) analysesrecovered trees of identical topology (Fig. 2),except for the unresolved position of Euglenatristella, see below. The tree shows only singlemodel Bayesian support (pp) values then the MLsupport (bp) values. When the Bayesian supportvalues differ between the single and partitionmodels, the single model values are given in thetext first, followed by the partition model values.

The backbone of this multi-gene tree was wellsupported by both Bayesian posterior probabil-ities (pp) and ML bootstrap numbers (bp). Withinthe ten ingroup genera ten separate clades (A–J),as well as a sister relationship of the single taxonEuglena proxima within the clades (A–G), were allwell supported. Each genus formed a single majorclade except for Euglena that formed two distinctwell-supported clades (A and G) and the singletaxon Euglena proxima making Euglena a poly-phyletic genus (Fig. 2). Consequently, Euglenariawas erected to contain the taxa of clade G.

Analysis of each dataset individually (nSSU,nLSU and cpSSU) each resulted in less well-resolved and supported topologies (not shown)with separate clades for each genus, as in thecombined tree. The nSSU tree was the mostsimilar to the combined tree and differed only inplacing the Euglenaria clade as sister to theEuglena clade (0.92 pp). However, the nLSU andcpSSU each supported the separation of Eugle-naria from Euglena, as did a combined nSSU andnLSU dataset (not shown). The combined nSSU/nLSU dataset resulted in a well-supported (pp)tree of identical topology to the three gene treepresented in this study (Fig. 2).

Overview

The phylogenetic relationships among the fresh-water euglenoid genera Euglena, Trachelomonas,

Figure 1. (A) Alignment of the nSSU sequences between helix 18 and 180. Euglenaria caudata and Euglenaviridis (representing all other euglenoids) are indicated with the base C among all members of the new genusEuglenaria and the base A among all other euglenoids used in this analysis, with the exception of Eutreptiaviridis which has a G in this position (not shown). (B) Alignment of the nSSU sequences between helix 37 and38. Euglenaria caudata and Euglena viridis (representing the genus Euglena) are indicated with the base Tamong all members of the new genus Euglenaria and the base A or C (not shown) among all members of thegenus Euglena used in this analysis. The diagnostic bases are in bold. (C) Alignment of nSSU sequences ofstem 17 between Euglena proxima and Euglena viridis (representing all other euglenoids). Euglena proximahad a unique CGC\CCG sequence while most other euglenoids had a CAA\TTG sequence. The bases in thestem of helix 17 are in bold.

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Strombomonas, Colacium, Monomorphina, Cryp-toglena, Euglenaria, Lepocinclis, Phacus, andDiscoplastis are represented in Figure 2. Theingroup taxa were split into two main clades.Clade one was well supported by both Bayesian

and ML analyses (pp 0.99 and 1.00, bp 84%) andcontained eight genera: Euglena, Trachelomonas,Strombomonas, Colacium, Monomorphina, Cryp-toglena, and Euglenaria, as well as the singletaxon Euglena proxima. Clade two was well

Figure 2. A rooted single model Bayesian (B) phylogenetic tree based on 5161 characters (nSSU, nLSU andcpSSU) obtained from 85 taxa. The Maximum Likelihood (ML) tree had identical topology. Numbers on theinternodes represent Bayesian single model posterior probability (pp)/ML bootstrap support for the node. Forpp values only those above 0.80 are shown. The n indicates a pp=1.00 or bootstrap=100%. Each taxon nameis followed by its SSU culture collection origin. The tree was outgroup rooted posteriorly with Eutreptia andEutreptiella species. Scale bar represents number of substitutions/site. The clades that contained eachgenus are indicated by the capital letter at nodes of the tree.

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supported (pp 0.96 and 0.99) by Bayesian, butweakly supported (bp 67%) by ML analysis,contained three genera: Lepocinclis, Phacus, andDiscoplastis.

Clades A–G

Euglena: After the transfer of five taxa to thenew genus Euglenaria (clade G), Euglena remainsparaphyletic. Euglena proxima was well supportedas an independent lineage from the crownEuglena clade A by Bayesian and ML analyses(pp 0.99 and 1.00, bp 84%) on its own branch.Euglena proxima lacked the diagnostic base C ofEuglenaria and instead had the base A as found inall the remaining ingroup taxa. Still, E. proxima hada unique molecular signature in the nSSU at thebase of stem 17 CGC\CCG where all but eighttaxa on the tree had CAA\TTG (Fig. 1C). The eighttaxa (seven ingroup and one outgroup) hadvarious differences in this stem area, but nonewere like those found in E. proxima.

The crown clade A (pp 1.00, bp 100%) containedall of the remaining members of Euglena includingthe type species, Euglena viridis. Nearly all sisterrelationships within clade A were well supportedby Bayesian posterior probabilities (0.96–1.00,0.99–1.00) with the majority supported by 1.00.The exceptions were the position of E. tristella assister to E. splendens weakly supported by ML(51%) but well supported by single Bayesian (0.96),but weakly supported as sister to E. sociabilisby partition Bayesian (0.61). Therefore, its finalposition is represented as an unresolvedtrichotomy. The position of E. splendens as sisterto E. stellata was well supported by Bayesian butonly weakly supported by ML (pp 1.00 and 0.99, bp63%). Neither Bayesian nor ML fully supported theposition of E. cantabrica and E. granulata as sisters(pp 0.65 and 0.83, bpo50%) or their sisterrelationships to the E. viridis, E. pseudoviridisclade (pp 0.82 and 0.85, bpo50%).

Trachelomonas/Strombomonas/Colacium:These sister clades (B, C and D) were connectedby a short branch that was well supported byBayesian analysis (pp 1.00 and 0.99), but onlyweakly supported by ML analysis (bp 59%). Allmembers of these three clades produced profuseamounts of mucilage that was retained on the cellsurface as either a lorica (Trachelomonas andStrombomonas) or a stalk (Colacium).

Relationships among members of Trachelomo-nas, a specious genus that formed various sizeand shaped loricas with spines and/or pores, were

all well supported (pp 1.00, bp 94–100%) andcontained within the single clade B (pp 1.00, bp100%). Clade B divided into three well supportedsister clades B1 (pp 1.00, bp 100%) B2 (pp 1.00,bp 99%) and B3 (pp 1.00, bp 100%), whichdiffered based on the morphology of their loricas.

All Strombomonas were contained in clade C(pp 1.00, bp 100%) and all sister relationshipsamong species were well supported (pp 1.00, bp100%). The sister relationship between Strombo-monas (clade C) and Trachelomonas (clade B) waswell supported (pp 1.00, bp 91%) as well.Colacium contained six described species withtwo present in the analysis. These two membersformed a single clade D (pp 1.00, bp 100%).

Monomorphina/Cryptoglena: The relationshipbetween these two sister clades was well sup-ported by both Bayesian and ML analysis (pp 1.00,bp 100%). Five members of Monomorphina werecontained within clade E (pp 1.00, bp 100%) withstrongly supported sister relationships (pp 1.00,bp 99–100%). The two members of Cryptoglenawere contained within clade F (pp 1.00, bp 100%).

Euglenaria: This newly erected genus containedfive taxa previously in Euglena (clade A). Molecularanalysis has shown that these taxa formed astrongly supported (pp 1.00, bp 100%) clade (G)separate from other Euglena species in cladeA and the single taxon Euglena proxima. The sisterrelationships among the members of thisnew genus were equally well supported (pp 1.00,bp 100%). The relationship of Euglenaria (clade G)to Monomorphina (clade E) and Cryptoglena (cladeF) was well supported by Bayesian (pp 1.00), butweakly supported by ML (bp 64%) analyses.

Clades H–J

Lepocinclis: Lepocinclis formed a single wellsupported clade H (pp 1.00, bp 100%). The sisterrelationships among the six species within thisclade were all strongly supported (pp 1.00, bp100%), except for the sister relationship betweenL. spirogyroides and L. acus that had only weakML support (bp 67%). The sister relationshipbetween Lepocinclis (clade H) and Phacus(clade I) was well supported (pp 1.00, bp 100%).

Phacus: Clade I was well supported by the singleBayesian model (pp 0.96) but had the weakestsupport of all the major clades (pp 0.82, bp 72%)from partition Bayesian and ML analyses. This cladecontained 15 members of Phacus, which includedthe new member P. limnophila. The placement of

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P. limnophila, was intriguing because of its spindle-shaped morphology, but was well supported (pp1.00, bp 96%) as a sister to P. warszewiczii (cellstriangular in apical view). Many of the branches inclade I were short with Bayesian and ML supportranging from no support (ppo0.90, bpo50%) to wellsupported (pp 1.00, bp 79%). The Phacus in clade I1were strongly supported (pp 1.00, bp 92%) as agroup, as were their sister relationships (pp 1.00, bp100%), except for P. ankylonoton (pp 0.89 and 0.84,bp 64%).

Discoplastis: This genus diverges prior to theLepocinclis and Phacus clades with strong Bayesian,but weak ML support (pp 0.96 and 0.99, bp 67%).The two members of this genus were well supported(pp 1.00, bp 100%) in clade J as sister taxa.

Taxonomic Revisions

Euglenaria Karnkowska, Linton et Kwiatowski,gen. nov.

Cellulae libere viventes, solitariae, cum unoflagello emergenti ubi natans, claviformes, fusi-formes vel cylindrico-fusiformes, metabolicae,angustatae postice, gradatim decrescentes incaudam acutam; chloroplasti parietales, lobati,pyrenoidum singulum continentes; pilei paramyla-cei utrinque chloroplasto insidentes; mucocystaenullae.Diagnosis

Cells free-living, solitary with one emergentflagellum when swimming; club-shaped, fusiformor cylindrically-fusiform, narrowing to the posteriorand tapering into a pointed tail-piece; metabolic,display euglenoid movement; parietal, lobedchloroplasts, each with a single pyrenoid accom-panied by bilateral paramylon caps located onboth sides of the chloroplast (diplopyrenoid);mucocysts absent.

Type species:Euglenaria caudata (Hubner) Karnkowska et

Linton, comb. nov.Basionym:Euglena caudata 1886. Hubner, E.F.W. Eugle-

naceen-Flora von Stralsund. Program Realgym-nasiums zu Stralsund. p. 13, Fig. 15.

Euglenaria caudata var. minor (Deflandre) Karn-kowska et Linton, comb. nov.

Basonym:Euglena caudata var. minor Deflandre, Bull. Soc.

Bot. de France. 71: 1119, Fig. 7, 1924.Euglenaria clavata (Skuja) Karnkowska et Linton,

comb. nov.

Basionym:Euglena clavata Skuja 1948. Taxonomie des

phytoplantons einigen Seen in Uppsala, Schwe-den. Symbolae Botanicae Upsaliensis 9(3): 189,190, pl. 22, Figs. 2–5.

Euglenaria anabaena (Mainx) Karnkowska etLinton, comb. nov.

Basionym:Euglena anabaena 1926. Mainx, F. Einige

neue Vertreter der Gattung Euglena Ehrenbergmit Unterstutzung der Gesellschaft zur Forderungdeutschen Wissenschaft, Kunst und Literaturin Bohmen. Arch. Protistenkd 54: 160-161,Fig. Dc.

Euglenaria anabaena var minima (Mainx)Karnkowska et Linton, comb. nov.

Basionym:Euglena anabaena var minima 1927. Mainx, F.

Beitrage zur Morphologie und Physiologie derEuglenen. I. Morphologische Beobachtung, Meth-ode, und Erfolge der Reinkultur. II. Untersuchunguber die Ernahrungs- und Reizphysiologie. Arch.Protistenkd 60:334, pl. 10, Figs. 3, 4.

The name, Euglenaria, was derived from thegenus Euglena and the Greek, ‘‘-aria’’ n. meaninglike. Thus, these taxa were morphologically indis-tinguishable from members of Euglena, but weregenetically distinct based on phylogeneticanalysis. Moreover, in the nSSU alignment allmembers of Euglenaria had a C base, four basesupstream of helix 180, where all other ingroupeuglenoids and the two Eutreptiella species havean A, while Eutreptia viridis has a G (Fig. 1A).Furthermore, in the nSSU alignment all membersof Euglenaria had a T, one base upstream of helix38, while all members of Euglena had either an Aor a C (Fig. 1B). The T base at this position wasfound in all other ingroup taxa except for Phacushamelii and P. segretti, which had the C base. Thebase C at this position was also found in all threeoutgroup taxa suggesting that C is the primitive(plesiomorphic) state. Together, the C baseupstream of helix 180 and the T base upstreamof helix 38 separate the Euglenaria from membersof Euglena and the other ingroup euglenoids.

PhacusThe genus Phacus was created and diagnosed

by Dujardin (1841) as rigid, free-swimming cellsthat were more or less flattened. Recently Marinet al. (2003) emended this diagnoses to moreaccurately define Phacus as a genus. However,due to Lepocinclis salina and Euglena limnophilabeing transferred into Phacus (see below), anemended diagnosis of the genus was againrequired.

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Phacus Dujardin, Historie naturelle des Zoo-phytes-Infusoires. Paris. pp. 327, 334, 1841.Emend. Linton et Karnkowska

Phacus – Emended DiagnosisCells free-living, solitary with a single emergent

flagellum when swimming; usually laterally com-pressed (rarely not compressed or triangular in apicalview), spindle-shaped or ovoid; semi-rigid to rigid;numerous parietal disc-shaped chloroplasts of similarsize without pyrenoids; paramylon grains dimorphicin size, large ring-shaped, rod-shaped or discoidparamylon grains. Colorless forms known.

Phacus salina (Fritsch) Linton et Karnkowska,comb. nov.

Basionym:Lepocinclis salina 1914. Fritsch, F.E. Notes on

British Flagellates, I-IV. The New Phytologist, vol.13, Iss. 10. pg. 351, Fig. 3A, B.

Synonyms:Lepocinclis texta var. salina (Fritch) Popova,

Trudy Botanicheskogo Instituta Akademii NaukSSSR. 7: 301, pl. 11 Fig. 18, 1951; Euglena textavar. salina (Fritch) Popova, Euglenovyje vodorosli.Opredelitel prosnovodnych vodoroslej SSSR, 7.[Euglenophyta. The Handbook of FreshwaterAlgae]. pg. 159, Fig. 59, 1955.

Phacus limnophila (Lemmermann) Linton etKarnkowska, comb. nov.

Basionym:Euglena limnophila Lemmermann, Beitrage zur

Kenntnis der Planktonalgen. II. Beschreibung neuerFormen. Beihefte Botan. Centralbl. 76: pg 152 andLemmermann in Pascher’s Sussw. – Fl., 2: 130,Fig. 205, 1913.

Phacus was distinguished from Lepocinclisbased on phylogenetic analysis and moleculardifferences present in the nSSU alignmentbetween the two genera. The base T waspresent one base downstream of helix 15 andtwo bases downstream of helix 230 in all Lepocin-clis whereas all Phacus had the base C and A,respectively.

Discussion

Euglena

Past studies of euglenoid phylogenies have usedonly nuclear encoded genes. Most often this wasthe nuclear SSU rDNA (Busse and Preisfeld2002, 2003; Busse et al. 2003; Leander et al.2001; Linton et al. 1999, 2000; Marin et al. 2003;Preisfeld et al. 2000, 2001; Von der Heyden et al.2004) with one study (Talke and Preisfeld 2002)using the nuclear encoded flagellar PAR1 and

PAR2 genes, the results from which agreed withprevious nuclear SSU rDNA studies. The first non-nuclear gene study used chloroplast SSU rDNAsequence data from 17 photosynthetic species(Milanowski et al. 2001) and suggested polyphylyin Euglena, but the bootstrap support was weak.

More recent molecular studies have combined twogenes. The first by Milanowski et al. (2006) combinednuclear and chloroplast SSU rDNA sequence datafrom 35 ingroup and 2 outgroup taxa to conclude,among other things, that Euglena was a polyphyleticgenus with members present within four separateclades (A, BþCþDþ, E and G) of their tree. Clade Acontained most members of Euglena including thetype specimen (E. viridis), BþCþDþ containedEuglena anabaena, E contained E. proxima while Gcontained Euglena spathirhyncha and E. limnophila.The second study by Triemer et al. (2006) combinednuclear SSU and partial LSU rDNA sequence data toestablish a well resolved and supported phylogeny ofEuglenales (sensu Leedale). Their analysis of 84 taxasupported 9 ingroup genera and established the newgenus Discoplastis to contain D. spathirhyncha andD. adunca, two former members of Euglena. How-ever, their study did not include E. proxima or E.limnophila as in Milanowski et al. (2006), but it didinclude Euglena anabaena and three sister taxa thatformed an ‘‘anabaena’’ clade. This ‘‘anabaena’’ cladewas supported (pp 0.92, Fig. 1 Triemer et al. 2006) assister to the other members of Euglena. In summary,Discoplastis was erected to contain some formermembers of Euglena (Triemer et al. 2006) andestablish the monophyly of the genus. Unfortunately,Milanowski et al. (2006) showed that at least threeadditional members of Euglena (E. proxima, E.limnophila and the ‘‘anabaena’’ clade) would requireeither the erection of a new genus or the transfer toan existing genus to fully establish the monophyly ofEuglena. By combining a larger number of taxa andthree genes, this study has resolved two of the threeremaining taxonomic issues necessary to establishthe monophyly of Euglena.

This study has shown that other than mostmembers having a spindle shaped body, noderived (synapomorphic) morphological or mole-cular character capable of uniting members ofEuglena has yet been identified. However, someimportant characteristics of this genus wereidentified. The chloroplasts show more morpho-logical diversity than any other genus, rangingfrom absent to ribbon-like to lenticular to lobed ordiscoid with pyrenoids present in the chloroplastof some species but absent in others. Moreoverthe mucocysts, mucus secreting bodies below thepellicle strips, show great morphological diversity.

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They may be absent or present, spherical orspindle in shape (Kosmala et al. 2009).

Because of the morphological diversity inEuglena, the species E. proxima (with chloroplaststhat are discoid and lack pyrenoids) cannot beseparated from Euglena (clade A) nor joined to theEuglenaria (clade G), because it lacks the diag-nostic nSSU bases (see diagnosis above). At thistime E. proxima must remain as a single unasso-ciated taxon until additional data (sequence and/or taxa) cause it to either clade with an existinggenus or other taxa clade with it allowing thediagnosis of a new genus.

Regrettably, despite the erection of Euglenaria(above) and the transfer of Euglena limnophila toPhacus the genus Euglena remains paraphyletic.Clade A contains the type Euglena viridis and thusretains the name Euglena.

Trachelomonas/Strombomonas/Colacium

A great deal of debate has occurred recently(Brosnan et al. 2003, 2005; Ciugulea et al. 2008;Marin et al. 2003; Moreira et al. 2001; Mullner et al.2001; Nudelman et al. 2003; Triemer et al. 2006)about the existence of Strombomonas (clade C)and whether to dissolve it back into Trachelomo-nas (clade B). While all of these molecular studiessupported the monophyly of the loricate forminggenera (Brosnan et al. 2003; Marin et al. 2003;Moreira et al. 2001; Mullner et al. 2001; Nudelmanet al. 2003) most failed to sustain Trachelomonasand Strombomonas as distinct genera (significantpp or bp support). All of these studies sufferedfrom limited taxa sampling, a weakness recog-nized by many of the authors (Brosnan et al. 2003;Moreira et al. 2001; Mullner et al. 2001; Nudelmanet al. 2003). Fortunately, the two most recentmolecular studies (Ciugulea et al. 2008; Triemeret al. 2006) used a larger number of taxa and twogenes (nuclear SSU/LSU rDNA), which showedsignificant support for maintaining these as dis-tinct genera.

The morphological diversity of the lorica hasbeen the chief diagnostic character essential forgenus and species identification within these twogenera. Our analysis has shown Trachelomonas tobe composed of three clades (B1, B2 and B3) thatcan be distinguished morphologically by the loricaand chloroplast. Species with spiny loricas, andlarge disc-shaped plastids with diplopyrenoids arecontained in clade B1, except for T. abrupta withinner projecting haplopyrenoids. Clade B2 con-tained species that are small and round with twoto five or more chloroplasts, five being the most

common, all with inner projecting haplopyrenoids.Clade B3 contained species that were oblong inshape with irregular surface ornamentation and upto ten chloroplasts, all with inner projectinghaplopyrenoids. Since all taxa in clades B2, B3and T. abrupta, the basal member of B1, containinner projecting haplopyrenoids; this would be theplesiomorphic state. These results are in agree-ment with the five Trachelomonas clades ofCiugulea et al. ((2008) A–E, Fig. 1).

The four members of Strombomonas formed asingle clade (C) that was well supported anddistinct from its sister Trachelomonas (clade B) inour study. The lorica lacks the distinctive spinesand pores associated with the loricas of Trache-lomonas. Although, our study contained only fourtaxa compared to the eight of Ciugulea et al.((2008) F–H, Fig. 1) with three taxa from their cladeF and one from their clade H (S. ovalis), our resultsare congruent with theirs. We expect future workinvolving molecular and morphological data toenable a much-needed revision of the currentTrachelomonas and Strombomonas taxonomybased on lorica morphology alone.

The two members of Colacium anchored theprofuse mucilage producing genera in our study.Although Colacium species do not produce loricasthey do produce copious amounts of mucilagefrom their anterior end. This mucilage is used forattachment to a substrate and subsequent stalk,and in some species, colony formation (Rosowskiand Kugrens 1973; Rosowski and Willey 1977).Moreover, like most Strombomonas and the basalmembers of Trachelomonas, all species of Cola-cium possess inner projecting haplopyrenoids.Our results are in agreement with recent molecularstudies (Ciugulea et al. 2008; Triemer et al. 2006)that support Colacium as the basal clade tocopious mucilage producing genera. Notably, thisis the first study to support the anchor position ofColacium by ML analysis, although the bootstrapsupport was weak (59%).

Monomorphina/Cryptoglena

Five members representing three species ofMonomorphina were used in our analyses. Allmembers of Monomorphina are diagnosed sensuKosmala et al. (2007) as being ovoid or slightlyoblate and rigid; a single parietal, sphericalchloroplast that is often perforated, 2–4 prominentlateral paramylon plates between the pellicle andthe chloroplast with M. pyrum as the epitype.Furthermore, based on molecular and morpholo-gical evidence Kosmala et al. (2007) synonymized

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11 species of Monomorphina as M. pyrum anderected one new species M. pseudopyrum for theculture CCAC 0093 (originally misidentified as M.reeuwykiana by Marin et al. 2003). Our studysupports their changes and we have followed theirrecommendations; M. inconspicuus (ACOI 1295)and M. ovata (SAG 1244-5) are now considered asM. pyrum. Additionally, our study supports Mono-morphina (clade E) as being monophyletic andsister to Cryptoglena (clade F).

The members of Cryptoglena are similar toMonomorphina; cells ovoid, rigid with a singleparietal chloroplast and lateral, shield-shapedparamylon grains, pellicle lacking strip reduction.However, these genera have been shown inprevious molecular studies (Ciugulea et al. 2008;Kosmala et al. 2007; Marin et al. 2003; Milanowskiet al. 2006; Triemer et al. 2006) to be closelyrelated, but distinct genera. Additionally, based onthe amended diagnosis (Kosmala et al. 2007)Cryptoglena differ by lacking the strong pellicularridges found in Monomorphina, possessing amedian longitudinal furrow and having a chlor-oplast in the form of an open cylinder (C-shaped)lacking pyrenoids. These traits distinguish Cryp-toglena from Monomorphina. Our study supportsthe sister relationship between these genera andthe sister relationship to the newly erected genusEuglenaria.

Euglenaria

Members of this genus were formerly placed in thegenus Euglena and were morphologically indis-tinguishable from members of that genus. Thisposition was supported by molecular analyses,such as Marin et al. (2003) and Triemer et al. (2006)as well. However, molecular analysis byMilanowski et al. (2001, 2006) suggested thatEuglena anabaena was not a member of Euglena.But these studies had only one member of what isnow the Euglenaria clade (G) Euglena anabaena,so no reassignment of the taxon could have beenmade. Our study included five taxa in clade G,which has allowed an evaluation of both themorphological and molecular evidence. Morpho-logically uniting features of Euglenaria arethe parietal lobed chloroplast with diplopyrenoidsand absence of mucocysts. However, thesemorphological features do not differentiationEuglenaria from Euglena. Instead, the molecularnSSU characters are the diagnostic charactersthat separate Euglenaria from Euglena. While allEuglenaria have a C base upstream of helix 180

and a T base upstream of helix 38, all Euglena

have an A base upstream of helix 180 and a C or Abase upstream of helix 38. Furthermore, bothBayesian and ML analyses supported the separa-tion of these five taxa from Euglena and theirplacement as sister to the Cryptoglena andMonomorphina clades. Therefore, there is strongevidence and support for the erection of the newgenus Euglenaria and the transfer of five formermembers of Euglena into it.

Lepocinclis/Phacus

Six members of Lepocinclis and 15 members ofPhacus were included in our study. Cells are rigidor semi-ridged, sometimes exhibiting a slightbending or twisting motion, but none are meta-bolic as defined by Triemer et al. (2006). The cellscontain numerous small discoid chloroplast thatlack pyrenoids. This diagnosis could be appliedequally to both Phacus and Lepocinclis. The majordistinguishing characteristic was that all membersof Phacus were laterally compressed or flattened,while all members of Lepocinclis were notflattened in cross section. However, our studyhas resulted in the transfer of Lepocinclis salinaand Euglena limnophila into the genus Phacusrequiring the diagnosis to be emended to includecells that are spindle-shaped and ovoid. There-fore, being flattened is no longer a definingcharacteristic separating Phacus from Lepocin-clis.

What evidence is there for continuing thisseparation and not dissolving Lepocinclis (Perty1849) into Phacus (Dujardin 1841)? Neither this norany previous molecular phylogenetic study hasshown merging between these two genera; allhave shown them to be separate, well supportedclades. In addition, we have defined molecularsynapomorphies (shared derived) that distinguishthese genera from each other. In the nSSUalignments Lepocinclis species have the nucleo-tide T one base downstream of helix 15 and twobases downstream of helix 230. At these samepositions in Phacus the nucleotides C and A arepresent, respectively. We therefore recognizethese as two closely related sister clades andrecommend maintaining them as separate distinctgenera.

Discoplastis

Erected by Triemer et al. (2006), the two membersof Discoplastis contain numerous small discoidchloroplasts lacking pyrenoids and cells that arenot flattened, characteristics found in the closely

611Euglenoid Evolution using Three Genes

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related Lepocinclis and Phacus genera. However,Discoplastis is easily distinguished from the othertwo genera by being strongly metabolic, acharacteristic not found in either Lepocinclis orPhacus. Our phylogenetic analysis of the mole-cular differences among these genera supportsthis genus and its basal position to the Lepocinclisand Phacus genera.

Our study was the first euglenoid phylogeny touse three genes, two nuclear and one chloroplastto establish relationships among freshwaterphotosynthetic euglenoids. This study showedstrong support by both Bayesian and ML analysisfor 10 major ingroup clades with 10 correspondinggenera and the single unallied taxon Euglenaproxima. Our analysis resulted in the transfer oftwo taxa (Lepocinclis salina and Euglena limno-phila) to the genus Phacus and the concomitantemended diagnosis of Phacus to contain cells thatwere not flattened. This represents the firstchange in the defining morphological character-istic, cell flattened, of Phacus since Dujardinestablished it in 1841. The polyphyletic genusEuglena was moved closer towards monophyly bythe erection and transfer of five taxa to the newgenus Euglenaria along with the transfer ofEuglena limnophila to the genus Phacus. Unfortu-nately the unallied, but strongly supported, posi-tion of Euglena proxima remains and consequentlymaintains Euglena as a paraphyletic genus.

Methods

Cultures: Table 1 lists the 82 taxa with sources and GenBankaccession numbers used in this study. Alternative name(s) inculture collections or GenBank for the taxa used in this study,due to misidentification or taxonomic changes, are also givenin Table 1. All taxa were obtained from culture collections orwere collected from small ponds in New Jersey (NJ) andMichigan (MI), USA. Cultures NJ and MI used in this study,which are not available from public culture collections, will bemade available upon request. Ponds were sampled with aplankton net (mesh size, 20 mm) and euglenoid cells isolatedby a Pasteur capillary pipette. Cells were brought into unialgalculture and checked periodically for eukaryotic contaminants.All isolates were grown in modified AF-6 medium (Watanabeand Hiroki 1997) and/or soil–water medium (medium 3c,Schlosser 1994). Cultures were maintained at 20–22 1C underconditions of a 14:10 light:dark cycle with approximately30 mmol photons m�2 s�1 provided from cool whitefluorescent tubes.

Specimen identification: Identifications of cultures wereconfirmed microscopically when received using a ZeissAxioskop 2 Plus microscope (Carl Zeiss Inc., Hallbergmoos,Germany) or a Nikon Eclipse E-600 microscope (Nikon, Tokyo,Japan) both equipped with differential interference contrast(DIC) optics. Images were captured with an AxioCam HRC(Hallbergmoos) or Nikon DX-1200 photomicrographic system

attached to the microscope, respectively. A representativespecies of each clade in the context of their phylogeneticrelationships is shown in Figure 3.

DNA extraction, amplification, sequencing, andsequence alignment: DNA extraction, PCR amplificationprocedures, purification, sequencing, and sequence align-ment were conducted as described by Brosnan et al. (2003).Total genomic DNA was isolated from cultures with theDNeasy Tissue Kit (Qiagen Co., Valencia, CA, USA; Cat#69504) using the animal tissues protocol. The nSSU, nLSUand cpSSU rDNA sequences were amplified in 20-mLreactions. Amplification was performed using; GeneAmpPCR System 9700 (Perkin Elmer Co., Norwalk, CT, USA), MJResearch PTC-0200 DNA Engine Gradient Thermo Cycler (MJResearch Inc., Waltham, MA, USA), Mastercycler Personaland Mastercycler Gradient (Eppendorf AG, Hamburg, Ger-many), see Table 2 for PCR programs used. The PCRproducts were sized on 1% agarose gels and then purifiedusing the MiniElute Gel Extraction Kit (Qiagen Co.; Cat#28606) or QIAEXII Gel Extraction Kit (Qiagen Co.;Cat#20051) according to the manufacturer’s protocol. Thepurified template was sequenced with internal primers forconserved regions (nSSU, Linton et al. 2000 and Table 3;nLSU, Brosnan et al. 2003; cpSSU Table 3) using either an ABI377, 3700, 3730 or 3730xl dye terminator sequencer (AppliedBiosystems, Foster City, CA, USA). Sequences wereassembled into contigs by the SeqMan program from theLASERGENE package (DnaStar, Madison, WI, USA) or usingthe Contig Assembly Program (CAP2) in the Genetic DataEnvironment (GDE 2.2) program (Smith et al. 1994). Thesequences for the nSSU, nLSU and cpSSU rDNAs werealigned by eye using the secondary structure of Euglenagracilis Klebs as a guide (Kjer 1995; Wuyts et al. 2001) in GDE2.2. The conserved areas of the genes were readily alignableacross taxa and were used for phylogenetic analyses. Areasthat could not be unambiguously aligned were excluded fromanalyses. All three gene sequences used in this study werederived from the same culture when possible (Table 1). Whennot possible due to culturing or sequencing difficultiesalternative sources were used, the taxon identitieswere confirmed microscopically and by sequencecomparisons among the first 700 bases of the nSSUsequences.

Phylogenetic analysis: The three genes resulted in acombined dataset of 5161 bases: 1740 from nSSU rDNA,2110 from nLSU rDNA and 1311 from cpSSU rDNA foranalysis. All analyses were done from within MacGDE 2.3. TheBayesian Information Criterion (BIC) analysis using MODELT-EST 3.7 (Posada and Crandall 1998) was used to determinethe best model for each dataset (nSSU, nLSU and cpSSU) foruse in partition Bayesian analysis while a single model basedon the BIC was determined for the combined data for use inBayesian and Maximum Likelihood (ML) analysis. Trees wererooted using Eutreptia viridis, Eutreptiella braarudii andEutreptiella pomquetensis as outgroup taxa. The three genedata set and tree (Fig. 2) have been deposited in TreeBASE,study accession number is S2655 and the matrix accessionnumber is M5103.

Bayesian analyses: The analyses were performed in Mr.Bayes 3.1.2 (Ronquist and Huelsenbeck 2003) using a singlemodel for the data or with an individual model for each of thethree data partitions. Parameters for the single model were asfollows (GTRþIþG): Prset revmatpr=dirichlet (1.49, 3.86, 1.63,0.65, 5.78, 1.00) statefreqpr=dirichlet (0.23, 0.24, 0.29, 0.23)shapepr=exponential (0.79) pinvarpr=fixed (0.30). Parametersfor the partitioned datasets were as follows (GTRþIþG): nSSU

E.W. Linton et al.612

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Ta

ble

1.

Taxa

used

with

culture

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llectio

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des.

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ga

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ank

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ber

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I107

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107

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999907

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999910

EU

624035

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mucro

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elly

et

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ud

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AJ532441

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999906

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221482

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140101

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221483

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skuja

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onia

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10.8

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014998

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130236

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221484

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140100

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626060

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750713

FJ486278

EU

750706

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ag

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115279

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624033

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cla

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nb

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613Euglenoid Evolution using Three Genes

Author's personal copy

Tab

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221494

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373

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221485

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ner)

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750714

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624010

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750698

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(Defland

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750708

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140108

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289247

Eutr

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249879

EU

624026

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249873

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750699

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152104

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130226

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221495

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935697

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624011

AF

289249

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110419

AY

130235

AY

221726

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spiro

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130814

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289243

(Eug

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523

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615Euglenoid Evolution using Three Genes

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Figure 3. A rooted Bayesian phylogenetic tree, as in Figure 2 but with each of the major clades collapsed(indicated by a capital letter) and a representative taxon from that clade pictured (not to scale). Names ofpictured taxa from left to right; Euglena viridis, Trachelomonas hispida, Strombomonas verrucosa, Colaciummucronatum, Monomorphina pyrum, Cryptoglena skujae, Euglenaria anabaena, Euglena proxima, Lepocinclisspirogyroides, Phacus orbicularis, Discoplastis spathirhyncha. The tree was outgroup rooted posteriorly withEutreptia and Eutreptiella species (not shown).

Table 2. PCR reaction parameters.

General PCR parameters96 1C 2 min, 35 cycles (95 1C 30 s, 37 1C – 56 1C 30 s, 72 1C 1 min) 72 1C 6 min, and a 4 1C hold

PCR parameters for cpSSUF/cpSSUR and cpSSU647F/cpSSUR95 1C 5 min, 37 cycles (95 1C 1 min, 44 1C 2 min, 72 1C 2 min – 7 cycles and 95 1C 30 s, 54 1C 30 s, 72 1C1 min – 30 cycles) 72 1C 10 min, and a 7 1C hold

PCR parameters for cpSSUF/cpSSU854R951 C 5 min, 37 cycles (951 C 1 min, 481 C 2 min, 721 C 2 min – 7 cycles and 951 C 30 s, 561 C 30 s, 721 C1 min – 30 cycles) 721 C 10 min, and a 71 C hold

PCR parameters for nSSUF/nSSU557R95 1C 5 min, 39 cycles (95 1C 1 min, 48 1C 90 s, 72 1C 1 min – 7 cycles and 95 1C 30 s, 56 1C 30 s, 72 1C 45 s– 32 cycles) 72 1C 10 min, and a 7 1C hold

PCR parameters for nSSU382F/nSSU1263R95 1C 5 min, 39 cycles (95 1C 1 min, 40 1C 90 s, 72 1C 90 s – 7 cycles and 95 1C 30 s, 49 1C 30 s, 72 1C 90 s –32 cycles) 72 1C 10 min, and a 7 1C hold

PCR parameters for nSSU1154F/nSSUR95 1C 5 min, 37 cycles (95 1C 1 min, 54 1C 1 min, 72 1C 45 s – 7 cycles and 95 1C 30 s, 59 1C 30 s, 72 1C 45 s– 32 cycles) 72 1C 10 min, and a 7 1C hold

E.W. Linton et al.616

Author's personal copy

revmatpr=dirichlet (1.37, 3.16, 1.45, 0.59, 4.53, 1.00) state-freqpr=dirichlet (0.21, 0.29, 0.28, 0.22) shapepr=exponential(0.73) pinvarpr=fixed (0.28); nLSU revmatpr=dirichlet (1.70,3.69, 1.80, 0.62, 6.23, 1.00) statefreqpr=dirichlet (0.21, 0.26,0.33, 0.21) shapepr=exponential (0.82) pinvarpr=fixed (0.33);cpSSU revmatpr=dirichlet (1.00, 5.18, 1.00, 1.00, 8.85, 1.00)statefreqp=dirichlet (0.33, 0.13, 0.20, 0.34) shapepr=expo-nential (0.59) pinvarp r=fixed (0.24). The analysis used fourMarkov chains (4,000,000 generations per chain), with treessaved every 100 generations discarding the first 8,000 trees. Amajority-rule consensus tree was created from the remaining32,001 trees. Convergence among these trees was confirmedvia the sump command.

Maximum likelihood analyses: The analysis was per-formed with PAUPn 4.0b10 for Macintosh OS X (Swofford 2002)using the following parameters (GTRþIþG): Base=(0.22750.2439 0.2943) Nst=6 Rmat=(1.4872 3.8611 1.6340 0.64815.7788) Rates=gamma Shape=0.7907 Pinvar=0.3016Shape=0.7907 NCat=4 RepRate=Mean Initbrlen=Rogers STart-vals=ParsApprox RECon=Marginal. The single ML tree wasobtained by a heuristic search using random stepwise additionwith 5 replicates, TBR branch swapping and MULTREES on.The 100 ML bootstrap repetitions were obtained by a Heuristicsearch using Random Stepwise addition with two replicates.The 100 non-parametric bootstraps were done as foursimultaneous independent searches of 25 bootstraps, eachrun on a separate CPU. The trees were aggregated andweighted according to the number of trees found in eachbootstrap replicate, so that all replicates had equal weight, a50% majority rule consensus bootstrap tree was obtained.

Acknowledgements

The authors wish to acknowledge the financialsupport provided by the National Science Foun-dation PEET program (Partnership for EnhancedExpertise in Taxonomy, grant no. DEB 4-21348)and by the Ministry of Science and HigherEducation grant no. N303 010 32/0552. We thankProf. Tomasz Majewski, Warsaw, Poland, forproviding a Latin diagnose for Euglenaria gen. nov.

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Primer Sequence (5’–3’)

cpSSU-F TTG ATC CTG GCT CAG GAT GAA CGC TcpSSU-647F ATT TCC AGT GTA GCG GTGcpSSU-854R AGG CGG GAC ACT TAA CGC GTTcpSSU-R CAA GGA GGT GAT CCA GCC GCA CCT TnSSU-F CAG TGG GTC TGT GAA TGG CTC CnSSU-557R TTA CCG CAG CTG CTG GCnSSU-570F GTG CCA GCA GCT GCG GTnSSU-1141F GAA ACT TAA AGG AAT TGnSSU-1154F GGA ATT GAC GGA ATG GCA CCnSSU-1263R GAG CGG CCA TGC ACC ACnSSU-R CGA CGG GCG GTG TGT ACA AGT

F=Forward Primer, R=Reverse Primer.

617Euglenoid Evolution using Three Genes

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619Euglenoid Evolution using Three Genes