Previous studies using the nuclear SSU rDNA have indicated that the photosynthetic euglenoids are a monophyletic group; however, some of the genera within the photosynthetic lineage are not monophyletic. To test these results further, evolutionary relationships among the photosynthetic genera were investigated by obtaining partial LSU nuclear rDNA sequences. Taxa from each of the external clades of the SSU rDNA-based phylogeny were chosen to create a combined dataset and to compare the individual LSU and SSU rDNA datasets. Conserved areas of the aligned sequences for both the LSU and SSU rDNA were used to generate parsimony, log-det, maximum-likelihood and Bayesian trees. The SSU and LSU rDNA consistently generated the same seven terminal clades; however, the relationship among those clades varied depending on the type of analysis and the dataset used. The combined dataset generated a more robust phylogeny, but the relationships among clades still varied. The addition of the LSU rDNA dataset to the euglenophyte phylogeny supports the view that the genera Euglena, Lepocinclis and Phacus are not monophyletic and substantiates the existence of several well-supported clades. A secondary structural model for the D2 region of the LSU rDNA was proposed on the basis of compensatory base changes found in the alignment.

The primary taxa of eukaryote classification should be monophyletic and based on fundamental cell structure rather than nutritional adaptive zones. The classical two kingdom classification into "plants" and "animals" and the newer four kingdom classifications into "protis","fungi""animals" and "plants" are therefore both unsatisfactory. Eukaryotes can be classified into nine kingdoms each defined in terms of a unique constellation of cell structures. Five kingdoms have plate-like mitochondrial cristae:(1) Eufungi (the non-ciliated fungi, which unlike the other eight kingdoms have unstacked Golgi cisternae),(2) Ciliofungi (the posteriorly ciliated fungi),(3) Animalia (Animals, sponges, mesozoa, and choanociliates; phagotrophs with basically posterior ciliation),(4) Biliphyta (Non-phagotrophic, phycobilisome-containing, algae; i.e. theGlaucophyceae and Rhodophyceae),(5) Viridiplantae (Non-phagotrophic green plants, with starch-containing plastids). Kingdom (6), the Euglenozoa, has disc-shaped cristae and an intraciliary dense rod and may be phagotrophic and/or phototrophic with plastids with three-membraned envelopes. Kingdom (7), the cryptophyta, has flattened tubular cristae, tubular mastigonemes on both cilia,m and starch in thecompartment between the plastid endoplasmic reticulum and the plastid envelope; their plastids, if present, have phycobilins inside the paired thylakoids and chlorophyll c2. Kingdom (8), the Chromophyta, has tubular cristae, together with tubular mastigonemes on one anterior cilum and/or a plastid endoplasmic reticulum and chlorophyll c1 + c2. Members of the ninth kingdom, the Protozoa, are mainly phagotrophic, and have tubular or vesicular cristae (or lack mitochondria altogether), and lack tubular mastigonemes on their (primitively anterior) cilia; plastids if present have three-envelop membranes, chlorophyll c2, and no internal starch, and a plastid endoplasmic reticulum is absent. Kingdoms 4-9 are primitively anteriorly biciliate. Detailed definitions of the new kingdoms and lists of the phyla comprising them are given. Advantages of the new system and its main phylogenetic implications are discussed. A simpler system of five kingdoms suitable for very elementary teaching is possible by grouping the photosynthetic and fungal kindoms in pairs. Various compromises are possible between the nine and five kingdoms systems; it is suggested that the best one for general scientific use is a system of seven kingdoms in which the Eufungi and Ciliofungi become subkingdoms of the Kingdom Fungi, and the Cryptophyta andChromophyta subkingdoms of th Kingdom Chromista; the Fungi, Viridiplantae, Biliphyta, and Chromista can be subject to the Botanical Code of Nomenclature, while the Zoological Code can govern the Kingdoms Animalia, Protozoa and Euglenozoa...

The flagellar apparatus of euglenoids consists of two functional basal bodies, three unequal microtubular roots subtending the reservoir, and a fourth band of microtubules nucleated from one of the flagellar roots and subtending the reservoir membrane. The flagellar apparatus of some euglenoids may contain additional basal bodies, striated roots ("rhizoplasts"), fibrous roots, striated connecting fibers between basal bodies, layered structures, or various electron-dense connective substances. With the possible exception of Petalomonas cantuscygni, nearly all euglenoids are biflagellate although the length of one flagellum may be highly reduced. The flagellar transition zone and number of basal bodies are highly variable among species. In recent years a cytoplasmic pocket that branches off from the reservoir has been discovered. The microtubules of the ventral flagellar root are continuous with the microtubules which line this pocket. Based on positional and structural similarities, this structure is believed to be homologous with the MTR/cytostome of bodonids. Coupled with other ultrastructural and biochemical data, the fine structure of the flagellar apparatus supports the belief that the euglenoid flagellates are descendant from bodonid ancestors.

Almost complete sequences of plastid SSU rDNA (16S rDNA) from 17 species belonging to the order Euglenales (sensu Nemeth, 1997; Shi et al., 1999) were determined and used to infer phylogenetic relationships between 10 species of Euglena, three of Phacus, and one of each of Colacium, Lepocinclis, Strombomonas, Trachelomonas and Eutreptia. The maximum-parsimony (MP), maximum-likelihood (ML) and distance analyses of the unambiguously aligned sequence fragments imply that the genus Euglena is not monophyletic. Parsimony and distance methods divide Euglenaceae into two sister groups. One comprises of representatives from the subgenera Phacus, Lepocinclis and Discoglena (sensu Zakryś, 1986), whereas the other includes members of Euglena and Calliglena subgenera (sensu Zakryś, 1986), intermixed with representatives of Colacium, Strombomonas and Trachelomonas. In all analyses subgenera Euglena--together with Euglena polymorpha (representative of the subgenus Calliglena)--and Discoglena--together with Phacus and Lepocinclis--form two well-defined clades. The data clearly indicate that a substantial revision of euglenoid systematics is very much required, nevertheless it must await while more information can be gathered, allowing resolution of outstanding relationships.

Phylogenetic analyses of 35 strains including 25 previously published sequences and 10 which have been newly sequenced, representing two species of Euglena, five species of Phacus and three species of Astasia, were carried out using the SSU rDNA. Parsimony, distance and maximum-likelihood inferred phylogenies support (1) monophyly of the euglenoids,(2) kinetoplastids as the sister group,(3) the phagotrophic Petalomonas cantuscygni Cann et Pennick anchoring the base of the euglenoid lineage,(4) evolution of phototrophy within the euglenoids from a single event,(5) multiple origins of osmotrophic euglenoids and (6) polyphyly of the genera Euglena Ehrenberg and Phacus Dujardin. Analyses also indicate that Lepocinclis Perty, Trachelomonas Ehrenberg and Astasia Dujardin are polyphyletic. In addition, the results suggest that neither the Euglenales nor the Eutreptiales form a monophyletic lineage, thus questioning currently available classifications. Concerning the phagotrophic mode of nutrition, the data suggest that the feeding apparatus arose multiple times.