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Systematics of primary osmotrophic euglenids: a molecular approach to the phylogeny of Distigma and Astasia (Euglenozoa)

Universität Bielefeld, Fakultät für Biologie, Postfach 100 131, 33501 Bielefeld, Germany

Correspondence
Angelika Preisfeld
a.preisfeld{at}uni-bielefeld.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Nuclear-encoded SSU rRNA genes from nine strains of Distigma and three strains of Astasia were sequenced and analysed phylogenetically with maximum-likelihood and maximum-parsimony methods. It could be demonstrated that the genus Distigma is paraphyletic, consisting of two distinct clades: one comprises four strains of the type species, Distigma proteus, and the other includes four strains of Distigma curvatum, Distigma gracile, Distigma sennii and Distigma elegans. These findings are well corroborated by morphological characteristics. The investigated species of Astasia are closely related to members of the Rhabdomonadida, thus rendering the genus Astasia polyphyletic, with Astasia longa branching within the phototrophs. All of the species investigated cluster in a well-supported group of primary osmotrophic euglenids that are not derived from photosynthetic ancestors. The recovered clades are characterized by their sequence diversity. After different evolutionary rates among lineages had been determined, a modified slow–fast approach was used to differentiate phylogenetic signal from noise. Finally, a revised systematic scheme based on phylogenetic relationships is suggested to render euglenid taxonomy more transparent: primary osmotrophic euglenids are classified as Aphagea, and members of the D. curvatum group are transferred into the new subgenus Parvonema.

Published online ahead of print on 16 August 2002 as DOI 10.1099/ijs.0.02295-0.

The GenBank/EMBL/DDBJ accession numbers for the SSU rDNA sequences from Distigma and Astasia species reported in this study are AF386637–AF386644, AF403152–AF403154 and AY061997, as detailed in Fig. 1.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Euglenids are a diverse group of protists that include phototrophic, phagotrophic and osmotrophic species. Phagotrophs are the base of the euglenid tree and probably represent the plesiomorphic mode of nutrition. Phototrophic forms emerged by means of secondary endocytobiosis: the chloroplast of an ingested green alga was established as the euglenid plastid. The origin of osmotrophic euglenids has been debated for a long time (e.g. Leedale, 1967, 1978; Dawson & Walne, 1994), as most of them lack taxonomically useful characteristics such as ingestion apparatuses or plastids. Use of molecular data (Linton et al., 1999, 2000; Preisfeld et al., 2000, 2001) and cladistic analyses of morphological characteristics of the euglenid pellicle (Leander et al., 2001) has revealed that only a fraction of the osmotrophic euglenids derived from phototrophic ancestors, while others originated from phagotrophs.

Members of the genus Distigma Ehrenberg emend. Pringsheim are characterized by two emergent flagella, a pronounced metaboly and the lack of ingestion devices, leading to a mode of nutrition supposed to be osmotrophic (Pringsheim, 1936, 1942; Skuja, 1948, 1956; Christen, 1959, 1962). This was supported by immunocytochemical results (Yamaguchi & Anderson, 1994), which located reaction products of acid phosphatase in small vesicles near the reservoir and canal region of Distigma proteus. This led to the idea of pinocytotic uptake of nutrients in this area. In his first description, Ehrenberg (1838) noticed two dark spots close to the base of the flagella, which he misinterpreted as eyespots, hence the name ‘Di-stigma’. It is now known that Distigma possesses neither an eyespot nor plastids (Yamaguchi & Anderson, 1994). De Fromentel (1874) was the first to propose a derivation of Distigma from the phototrophic genera Eutreptia and Eutreptiella by the loss of plastids, which is analogous to the proposed evolution of Astasia longa from Euglena gracilis-like ancestors (Siemeister & Hachtel, 1989).

Angeler and colleagues pointed out several features that are taxonomically important for the genus Distigma (Angeler, 1999, 2000; Angeler et al., 1999). These include the proportional length of the ventral flagellum in comparison with the dorsal one, the appearance of nuclear endosomes and the ultrastructure of the pellicle. D. proteus was found to possess symbiotic bacteria in its cytoplasm; because they were obviously not digested, it could be deduced that Distigma is derived from phagotrophic ancestors (Yamaguchi & Anderson, 1994; Angeler et al., 1999).

Early molecular investigations showed that D. proteus and Distigma curvatum are closely related to members of the Rhabdomonadida, thus constituting a well-supported clade composed of primary osmotrophic euglenids (Preisfeld et al., 2000, 2001). Furthermore, two Astasia species were found to branch within the primary osmotrophs rather than being closely related to the phototrophs (Linton et al., 1999, 2000; Müllner et al., 2001). Cladistic analyses of morphological characteristics confirmed the monophyly of primary osmotrophs and their divergence from phagotrophic ancestors with a flexible pellicle capable of euglenid movement (Leander et al., 2001).

The aim of this study was to investigate the phylogeny of Distigma, based on an SSU rDNA dataset. For this purpose, 12 new sequences from various Distigma and Astasia species were added to published sequences and the resulting dataset was critically analysed. Some taxonomically important morphological features were compared with the results based on molecular data, leading to the suggestion of a revised euglenid taxonomy.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Organisms, DNA extraction, amplification and sequencing.
Cultures of Distigma and Astasia species were obtained from the Culture Collection of Algae at the University of Göttingen, Germany (SAG), and the Culture Collection of Algae and Protozoa (CCAP), Ambleside, UK. The organisms were grown according to the culture-collection protocols. Total cell DNA was extracted using the DNeasy plant DNA purification kit (Qiagen). Amplification of nearly complete SSU rDNA was performed with universal eukaryotic primers by following the PCR protocols described previously (Preisfeld et al., 2000). PCR products were cloned into the pCR 2.1 vector using the TOPO TA cloning kit (Invitrogen). Sequencing was conducted with standard M13 primers as well as additional primers in both directions (primer walking).

Phylogenetic analyses.
Twelve new sequences from the genera Distigma and Astasia were aligned with published euglenozoan sequences. Initial alignments with CLUSTAL X gave only a rough estimate of positional homology. Consequently, the alignment was corrected manually, taking into account secondary-structure information from various organisms. Only unambiguously homologous positions were retained for phylogenetic inference.

Prior to maximum-likelihood (ML) tree inference, the best-fit model of sequence evolution was determined using MODELTEST 3.06 (Posada & Crandall, 1998). ML tree inference was undertaken using PAUP*, version 4.0b8 (Swofford, 1998); heuristic searches applying random addition of sequences and 10 replications have been conducted. ML bootstrapping was performed using a neighbour-joining tree as the starting topology and running 7500 rearrangements on each of the 100 replicates. Maximum-parsimony (MP) bootstrapping was done with random addition of sequences and 10 replications on each of the 500 bootstrap pseudosamples.

The Shimodaira–Hasegawa test, as implemented in PAUP*, was used to test for significant differences in likelihood among the ML tree and constrained topologies.

To characterize several monophyletic groups, revealed after phylogenetic analyses, the mean sequence diversity and standard deviation for selected groups were plotted. For this purpose, the pairwise ML distances, according to the optimal tree under the selected model of sequence evolution, were used.

A likelihood ratio test was performed with MODELTEST 3.06 to investigate the appropriateness of a molecular clock assumption. Therefore, the score of the optimal ML tree constrained to a molecular clock was calculated and compared with the score of the best tree without a clock assumption.

A relative-rate test implemented in PHYLTEST 2.0 (Kumar, 1996) was used to test the hypothesis of rate constancy among lineages. For distance calculation, Kimura's two-parameter model of sequence evolution (Kimura, 1980) with gamma distribution (alpha=0·8) was used. The sequences were arranged into groups and thus, different lineages could be compared, instead of single sequences.

In an attempt to differentiate phylogenetic signal from noise, and to deal with a non-stationary evolutionary rate among sites and taxa, a modified slow–fast approach according to Brinkmann & Philippe (1999) was performed. On the basis of an MP tree, the characteristics were grouped into categories according to the number of tree steps required at each position. This procedure allows for analyses of datasets that contain only a subset of the variable positions and therefore different proportions of homoplasy. Decay support for monophyletic groups found in parsimony analyses was determined with PAUP*, using the clade constraint method described by Morgan (1997).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
SSU rRNA-encoding regions from 12 euglenid species of the genera Distigma and Astasia were sequenced and analysed. The sequences were added to a euglenozoan dataset comprising euglenids, diplonemids, kinetoplastids and outgroups. The alignment contained 1141 characters, of which 617 were parsimony-informative.

After ML tree inference, the newly analysed sequences cluster in a well-supported clade of primary osmotrophic euglenids, including the genus Distigma, some species of the genus Astasia and the Rhabdomonadida (Fig. 1). The primary osmotrophs form the sister taxon to the phototrophs (including A. longa) and the phagotroph Peranema trichophorum. Monophyly of euglenids, including the phagotroph Petalomonas cantuscygni as first descendant, is weakly supported.

View larger version (78K): [in this window] [in a new window]   Fig. 1. ML tree of the Euglenozoa based on SSU rDNA sequences [symmetrical model according to Zharkikh (1994), assuming equal base frequencies and taking into account rate heterogeneity (alpha=0·8094) and invariable sites (pinvar=0·2016)]. Primary osmotrophic taxa are shown in bold. New sequences presented in this study are marked with asterisks. Numbers above branches indicate bootstrap proportions according to ML, and values below branches correspond to MP bootstrapping [where two numbers are separated by a solidus (/), they represent the ML/MP values]. Score of the best tree: -ln=15101·68.

The closest relatives of the D. curvatum group are represented by a strongly supported clade consisting of the monophyletic Rhabdomonadida and five strains of the genus Astasia, which do not form a monophyletic group.

By constraining the ML analyses, several user-defined trees were generated to address the significance of some hypotheses regarding the phylogeny of osmotrophic euglenids by Shimodaira–Hasegawa tests (Table 1). Constraint trees showing the monophyly of the Eutreptiina sensu Leedale (1967), combining the phototrophic genera Eutreptia and Eutreptiella as well as the osmotrophic genus Distigma, are significantly worse. Similarly, a monophyletic genus Astasia is rejected. The monophyly of the four analysed strains of D. curvatum is significantly dismissed. Although the sister-group relationship between the Astasia torta group and the Rhabdomonadida is supported with 100 % bootstrapping, the Shimodaira–Hasegawa test does not significantly reject the monophyly of the primary osmotrophic Astasia species.

View this table: [in this window] [in a new window]   Table 1. Shimodaira–Hasegawa tests comparing the ML tree shown in Fig. 1 with user-defined trees

View larger version (27K): [in this window] [in a new window]   Fig. 2. Sequence divergence plot. Means and standard deviations of pairwise ML distances according to the best-fit model of sequence evolution for selected groups. ‘AC+AT+R’, Monophyletic group comprising A. curvata, the A. torta group and the Rhabdomonadida; ‘Phototrophs’, phototrophic euglenids including A. longa.

The marked sequence diversity and the differences in evolutionary rate hint at the possibility that the sequences of the primary osmotrophs suffer from long-branch-attraction phenomena. In an attempt to deal with rate variation among lineages and sites and to distinguish noise from phylogenetic signal, a modified slow–fast approach was undertaken. The new datasets contained different categories of variable sequence positions measured as tree steps in the most parsimonious tree. Positions that change once over the tree topology are completely free of homoplasy and support well-known groups such as phototrophic euglenids, Rhabdomonadida, diplonemids and kinetoplastids (Fig. 3a). This dataset also supports the monophyly of the primary osmotrophic euglenids as well as most of the subgroups recognized before. As more variable sequence positions were added, the consistency index decreased and the signal-to-noise ratio was lowered. During the slow–fast procedure, the Bremer support for the primary osmotrophic clade and most of its subtrees increases continuously (Fig. 3b). Some clades revealed by ML analysis, such as the A. torta group or the entire euglenid group, are not supported at any step in the slow–fast approach.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Modified slow–fast approach. Support values are given as Bremer indices. (a) Most parsimonious tree based on onefold variable sequence positions: 63 informative sites, tree-length 63 steps, consistency index=1·00. (b) Strict consensus of five equally parsimonious trees based on the complete dataset containing all categories of positional variability: 617 informative sites, tree-length 3012 steps, consistency index=0·40. The MP branch length was computed with PAUP* (Swofford, 1998). The group of phototrophic euglenids includes A. longa.

Signature sequences
In addition to high bootstrap support and low genetic diversity implied by the SSU rDNA data, the D. curvatum group can be characterized by highly conserved sequence motifs (Fig. 4). These signature sequences are located within variable regions V2 and V4, surrounded by hypervariable areas for which no homologous positions could be identified either within this group or among euglenids. Although these motifs are conserved among the D. curvatum group, no homologous positions could be found in D. proteus or in the remaining members of the Euglenozoa. These complex nucleotide patterns can be considered an antapomorphy of the D. curvatum group, supporting their monophyly and their distinctiveness from D. proteus.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Signature sequences characterizing the D. curvatum group. Positions of signature sequences are given by reference to the SSU rDNA sequence of D. sennii. Accession numbers of sequences are given. Question marks in the sequence of D. proteus indicate that no homologous areas could be identified within this sequence. Signatures 1 and 2 are located in V2 and signature 3 in V4.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The D. curvatum group includes four strains of D. curvatum, D. gracile strain CCAP 1216/2, D. elegans and D. sennii. In contrast to the D. proteus strains, the ML tree topology and marked genetic distances indicate that D. curvatum comprises more than a single species. For instance, the species descriptions of D. curvatum (Pringsheim, 1936), D. gracile (Pringsheim, 1942) and Distigma glabrum (Christen, 1958; formerly assigned to D. curvatum SAG 1216-5) are very similar, as already stated by the respective authors. As distinctive features, they identified cell size and shape, characteristics which are to be considered insufficient for species diagnoses. Further morphological and ultrastructural investigations will be necessary to evaluate the species status of these strains and the validity of some of the diagnoses. Although some authors have proposed a close relationship to D. proteus (e.g. Christen, 1959), both D. sennii and D. elegans branch unambiguously within the D. curvatum group according to our molecular analysis. Moreover, D. sennii and D. elegans are not as closely related as postulated previously (Angeler et al., 1999; Angeler, 2000). These species are distinguished by different numbers of subpellicular microtubules, which may be responsible for the different capacity for metabolic movement (Angeler et al., 1999). However, a well-supported branching order within the D. curvatum group could not be achieved.

Morphologically, the genus Distigma is based mainly on plesiomorphic characteristics such as two emergent flagella, a flexible pellicle and a lack of chloroplasts. The splitting of Distigma into two distinct groups in molecular analyses (Preisfeld et al., 2000, 2001) is substantiated by morphological and ultrastructural characteristics: D. proteus possesses two flagella of equal thickness, the shorter flagellum reaching one-third of the body length. The nucleus contains up to three nucleoli, pellicle strips appear sigmoidal in transverse section and the surface is not covered by organic deposits (Angeler et al., 1999; Leander et al., 2001). In contrast, members of the D. curvatum group show a strongly reduced ventral flagellum and only one nucleolus per nucleus, and the pellicle is covered by a marked layer of organic deposit. Hence, we propose to create a new subgenus, Parvonema subgen. nov., comprising the species of the D. curvatum group.

The genus Astasia
The molecular data place the species of Astasia investigated in close affinity with the Rhabdomonadida, as shown previously by Müllner et al. (2001) using a smaller dataset. In contrast to A. longa (Linton et al., 1999, 2000), these species are not derived from phototrophic ancestors and thus belong to the primary osmotrophs. This ‘diphyletic origin’ of the genus Astasia was discussed previously by Leedale (1978). One reason for the polyphyly of the genus Astasia may be the imprecise circumscription, as it includes colourless uniflagellate euglenids capable of shape change (Pringsheim, 1942), neglecting the phylogenetic origin of osmotrophy. It could be speculated that additional species of Astasia will also show a close relationship to the Rhabdomonadida. Consequently, we suggest the transfer of secondarily osmotrophic Astasia species, such as A. longa, to Euglena. Since only three primary osmotrophic Astasia species were studied molecularly and no ultrastructural data are available, we informally term this assemblage ‘Euastasia’, following Müllner et al. (2001). Furthermore, the unavailability of the type species Astasia limpida Dujardin hinders taxonomic revision of the genus.

Primary osmotrophic euglenids
The euglenid primary osmotrophic clade has been recognized since molecular SSU rDNA data were used for phylogenetic reconstruction (Preisfeld et al., 2000, 2001; Frantz et al., 2000; Müllner et al., 2001). Our analyses corroborate the hypothesis that these organisms form an independent lineage within the euglenids without any phototrophic ancestor. Instead, primary osmotrophic euglenids are likely to be derived from phagotrophic ancestors (Leander et al., 2001). Correspondingly, the occurrence of cytoplasmic endobacteria in D. proteus (Yamaguchi & Anderson, 1994) could be ascribed to undigested prey of a phagotrophic euglenid.

A common feature of primary osmotrophs is an accelerated evolutionary rate, detectable in SSU rDNA sequences, accompanied by drastically enlarged genes encoding the small ribosomal subunit (Busse & Preisfeld, 2002b). As could be shown by several approaches, this characteristic does not interfere with phylogeny reconstruction by masking phylogenetic signal or long-branch-attraction effects.

Morphologically, the primary osmotrophs are combined because of negative characteristics such as the absence of plastids and ingestion devices. A putative synapomorphy might be the possession of split-ringed structures around the canal, as described for D. proteus (Leander et al., 2001), and which potentially represents a homologue of the scroll of the Rhabdomonadida (Leedale, 1967; Leedale & Hibberd, 1974). Additionally, primary osmotrophs share a common habitat, as all species were found in freshwater (Huber-Pestalozzi, 1955). On the basis of morphological and ultrastructural data, some noticeable evolutionary trends within this clade can be revealed (Leander et al., 2001). (i) The length of the ventral flagellum is continuously reduced: D. proteus possesses a ventral flagellum that reaches nearly one-third of the body length. In the D. curvatum group, it is further reduced to a short stub in D. sennii, whereas no ventral flagellum is emergent in A. curvata, A. torta or the Rhabdomonadida. (ii) The rigidity of the pellicle increases: whereas the pellicle of D. proteus is flexible, D. sennii is only moderately capable of euglenid metaboly. All members of the Rhabdomonadida are completely rigid, with a massive epiplasmic layer. This is correlated with a change in the appearance of the pellicle strips: they change from sigmoidal in D. proteus to flat in the Rhabdomonadida, in which they appear to be fused.

Euglenid taxonomy
Since the results presented have an impact on the understanding of euglenid phylogeny, we suggest a revision of parts of the existing taxonomic scheme based on the identification and naming of monophyletic taxa.

Several studies have shown the unambiguous monophyly of the primary osmotrophic euglenids (Busse & Preisfeld, 2002a; Preisfeld et al., 2000, 2001; Müllner et al., 2001). Consequently, we redefine the Aphagea [International Code of Zoological Nomenclature (ICZN); Aphagophycidae International Code of Botanical Nomenclature (ICBN)] of Cavalier-Smith (1993) to include solely primary osmotrophic euglenids that lack any vestiges of ingestion devices. Phototrophic euglenids, which have been a part of Cavalier-Smith's Aphagea, have been classified as Euglenea (ICZN; Euglenophycidae ICBN).

The suborder Eutreptiina Leedale (ICZN; Eutreptiales ICBN) has been emended, now comprising exclusively the phototrophic genera with two or more emergent flagella: Eutreptia, Eutreptiella and Tetreutreptia.

The Rhabdomonadida and the primary osmotrophic Astasia species have been included within the Rhabdomonadidia (ICZN; Rhabdomonadideae ICBN). Although several distinctive features of the D. curvatum group could be revealed, no unambiguous synapomorphy could be shown to link this group to the Rhabdomonadidia, thus supporting the paraphyly of Distigma. Consequently, species of the D. curvatum group are transferred into the new subgenus Parvonema.

Diagnoses
Diagnoses for new taxa are given according to the ICZN and the ICBN, following the approaches of several authors regarding the ambiregnal taxonomic status of protist taxa such as the euglenids (Patterson & Larsen, 1991, 1992; Larsen & Patterson, 1990; Novarino & Lucas, 1993, 1995).

Diagnosis of Aphagea Cavalier-Smith 1993 emend. Busse et Preisfeld (ICZN); Aphagophycidae subclassis nov. (ICBN)
Osmotrophic euglenids lacking photosensory apparatus and plastids; one or two emergent flagella; no ingestion apparatus. Composition: Rhabdomonadidia Busse et Preisfeld and Distigma Ehrenberg (ICZN); Rhabdomonadideae Busse et Preisfeld and Distigma Ehrenberg (ICBN).

Latin diagnosis of Aphagophycidae subclassis nov.
Euglenophyceae osmotrophicae; sine apparatu photosensorico; sine plastis; cum uno vel duobus flagellis emergentis; sine apparatu ingestione.

Diagnosis of Rhabdomonadidia subclassis nov. (ICZN); Rhabdomonadideae superord. nov. (ICBN)
Osmotrophic euglenids with one emergent flagellum; pellicle rigid or flexible. Composition: Rhabdomonadida Leedale 1967 and Astasia (ICZN); Rhabdomonadales Leedale 1967 and Astasia (ICBN).

Latin diagnosis of Rhabdomonadideae superord. nov.
Euglenophyceae osmotrophicae cum uno flagello emergente; pellicula rigida vel metabolica.

Diagnosis of Parvonema subgen. nov. (ICZN); Parvonema subgen. nov. (ICBN)
Primary osmotrophic euglenids with two emergent flagella. Ventral flagellum more reduced than in D. proteus Ehrenberg; pellicle strips flat with deposited organic material; no endobacteria, one nucleolus per nucleus. Type species: Distigma sennii Pringsheim 1942.

Latin diagnosis of Parvonema subgen. nov.
Euglenophyceae osmotrophicae; flagella duo, ambo ex canale emergentia; flagellum ventrale magis reductum quam apud Distigma proteus Ehrenberg; planae lineae pelliculae cum materia organica; sine endobacteria; nucleus cum uno nucleolo.

Diagnosis of Euglenea classis nov. (ICZN); Euglenophycidae subclassis nov. (ICBN)
Phototrophic euglenids with one or two emergent flagella; osmotrophic euglenids with photosensory apparatus and/or vestigial plastids; ingestion apparatus, if present, of the MTR-pocket type. Composition: Suborder Euglenina Leedale 1967 and Suborder Eutreptiina Leedale 1967 emend. Busse et Preisfeld (ICZN); Order Euglenales Leedale 1967 and Order Eutreptiales Leedale 1967 emend. Busse et Preisfeld (ICBN).

Latin diagnosis of Euglenophycidae subclassis nov.
Euglenophyceae phototrophicae cum uno vel duobus flagellis emergentibus; Euglenophyceae osmotrophicae cum apparatu photosensorico et/vel proplasto; apparatus ingestionis typo ‘MTR-pocket’.

Diagnosis of Eutreptiina Leedale 1967 emend. Busse et Preisfeld (ICZN); Eutreptiales Leedale 1967 emend. Busse et Preisfeld (ICBN)
Phototrophic euglenids with two or more emergent flagella and flexible pellicle; eyespot present. Composition: Eutreptia, Eutreptiella and Tetreutreptia.

Latin diagnosis of Eutreptiales Leedale 1967 emend. Busse et Preisfeld
Euglenophyceae phototrophicae cum duobus vel amplius flagellis emergentibus; cellulae metabolicae; cum stigma.

	ACKNOWLEDGEMENTS

 
We are very grateful to J.-W. Wägele for helpful discussions concerning taxonomy, and to B. Müller, Susanne Talke and Ulrike Brommund for reading the manuscript. This work was supported by the DFG (PR 624/2-1).

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