EVOLUTION OF PHACUS (EUGLENOPHYCEAE) AS INFERRED FROM PELLICLE MORPHOLOGY AND SSU rDNA

This research integrates a large morphological data set into a molecular context. Nineteen pellicle characters and 62 states from 13 euglenid taxa were analyzed cladistically. The pellicle morphology of Euglena tripteris (Klebs), Lepocinclis ovata (Conrad), Phacus brachykentron (Pochmann), P. oscillans (Klebs), P. pyrum (Stein), and P. triqueter (Dujardin) is described comprehensively. These data are compared with new information on the pellicle morphology of Euglena acus (Ehrenberg), E. stellata (Mainx), and Peranema trichophorum (Stein) in addition to published data on Entosiphon sulcatum (Dujardin), Euglena gracilis (Klebs), Distigma proteus (Pringsheim), and Petalomonas cantuscygni (Cann and Pennick). Nuclear small subunit (SSU) rDNA sequences provided an independent test for establishing a robust organismal pedigree of the same taxa. A synthetic tree derived from the combined phylogenetic analyses of pellicle morphology and SSU rDNA enabled us to parsimoniously map morphological character states. This approach demonstrated the utility of pellicle morphology for inferring phylogenetic relationships of euglenids and establishing apomorphy-based clade definitions. Three robust clades with unambiguous pellicle-based apomorphies can be recognized within taxa traditionally classified as Phacus : (1) L. ovata and P. pyrum , (2) E. tripteris and P. triqueter , and (3) P. brachykentron and P. oscillans. Taxonomic concerns that emerged from these results are discussed.     

PHYLOGENETIC AND TAXONOMIC POSITION OF LEPOCINCLIS FUSCA COMB. NOV. (=EUGLENA FUSCA) (EUGLENACEAE): MORPHOLOGICAL AND MOLECULAR JUSTIFICATION1

We studied the morphological diversity and analyzed the small subunit rDNA sequences of two taxa formerly known as Euglena spirogyra Ehr. and Euglena fusca (Klebs) Lemmermann. Our studies confirmed that the two should have the rank of a species, namely Lepocinclis spirogyroides (Ehr.) Marin et Melkonian and Lepocinclis fusca (Klebs) Kosmala et Zakry? comb. nov. (Euglenophyceae). We are defining new diagnostic features for these species, namely the size and the shape of the cells and the shape of the papillae, as well as designating epitypes for them.     

Comparative morphology of the euglenid pellicle. II. Diversity of strip substructure

The morphological diversity associated with the strip substructure of the euglenid pellicle was examined, and after identifying characters and states, we outlined hypotheses about their evolution. We have attempted to standardize terms necessary for analytical comparisons of strips by providing a glossary and comparing published synonyms. Most of the substructural diversity found in euglenids is demonstrated with 13 representative taxa. Strips are generally composed of two subcomponents: frames and projections. Frames support the basic shape of strips and many can be described as either S-shaped, plateau-shaped, M-shaped, or A-shaped. Projections branch laterally from the frames, are usually periodic, and can be described as thread-like structures, an indented plate, tooth-like structures, and plate-like structures. The ancestral state included strips that were few in number, flat, and fused. The strips became S-shaped and disjoined in the lineage leading to most euglenid taxa. These strips became secondarily flattened and fused in one lineage. In some lineages of phototrophs, the strips became increasingly robust. Two strips of different morphology formed the repeating pellicular unit or doublet in four taxa. These doublets evolved convergently at least three times and may provide insights into developmental patterns of the cytoskeleton.     

ARE CYTOPLASMIC POCKETS (MTR/POCKET) PRESENT IN ALL PHOTOSYNTHETIC EUGLENOID GENERA?1

In 1985, the existence of a cytoplasmic pocket formed from the reservoir membrane in the photosynthetic euglenoid Colacium was described. A band of reinforcing microtubules (MTR) derived from the ventral flagellar root lined the pocket, and a dense fibrillar mesh was associated with the membrane. A comparison of bodonid cytostomes, colorless euglenoid cytostomes, and the reservoir pocket found in Colacium suggested that the three structures were homologous and that photosynthetic euglenoids arose from phagotrophic ancestors. MTR/pockets have since been reported in other photosynthetic euglenoids, including Euglena, Eutreptia, Eutreptiella, Cryptoglena, Tetreutreptia, and Phacus. We found MTR/pockets in three additional taxa, Lepocinclis, Trachelomonas, and Strombomonas, thereby demonstrating the presence of this complex in representatives of all the major photosynthetic genera. A comparison of the MTR/pocket complex across genera indicated a reduction in structural complexity that was consistent with recent phylogenetic schemes based on molecular characters. Three alternative hypotheses of the origin of MTR/pockets in phototrophic euglenoids are presented and discussed.     

PHYLOGENY OF PHOTOSYNTHETIC EUGLENOPHYTES BASED ON COMBINED CHLOROPLAST AND CYTOPLASMIC SSU RDNA SEQUENCE ANALYSIS1

Eighteen new 16S rDNA and 16 new 18S rDNA sequences from 24 strains, representing 23 species of photoautotrophic euglenoids, were obtained in nearly their entire length. Maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses were performed on separate data (39 sequences of 16S rDNA and 58 sequences of 18S rDNA), as well as on combined data sets (37 sequences). All methods of sequence analysis gave similar results in those cases in which the clades received substantial support. However, the combined data set produced several additional well‐supported clades, not encountered before in the analyses of green euglenoids. There are three main well‐defined clades (A, B/C/D, and G) on trees from the combined data set. Clade G diverges first, while clades A and B/C/D form sister groups. Clade A consists of Euglena species sensu stricto and is divided into three sub‐clades (A1, A2, and A3). Clade A3 (composed of E. deses and E. mutabilis) branches off first; then, two sister clades emerge: A1 (composed of E. viridis‐like species) and A2 (consisting of E. agilis and E. gracilis species). Clade B/C/D consists of the Strombomonas, Trachelomonas, Cryptoglena, Monomorphina, and Colacium genera. Clade G comprises Phacus and Lepocinclis, as well as the Discoglena species of Euglena, with Discoglena branching off first, and then Phacus and Lepocinclis emerging as sister groups.     

A MOLECULAR ANALYSIS OF THE EUGLENOPHYTES USING SSU RDNA

Almost since the creation of the genus Euglena (Ehrenberg), the taxa assigned to it have been separated, split apart, and reorganized into new genera based on morphological relationships, resulting in the creation of the genera Phacus (Dujardin) , Lepocinclis (Perty) , Astasia (Pringsheim), and Khawkinea ( Jahn and McKibben) based on intuitive methods. In an effort to assess the validity of these genera, we have used small subunit (SSU) rDNA data to generate a phylogenetic framework for these genera, with particular attention to the genus Euglena . Using the conserved sequence areas, we performed a phylogenetic analysis using parsimony, maximum likelihood, and distance methods. These different criteria have resulted in trees of the same topology. The euglenoid clade was composed of phagotrophic euglenoids at the base, which gave rise to phototrophs that in turn gave rise to osmotrophs. Among the photosynthetic taxa, the biflagellate form diverged prior to the uniflagellate form. Additionally, the need for a revision in the taxonomy of some of these genera was demonstrated. Currently, taxa from the photosynthetic genera Euglena, Phacus, and Lepocinclis do not form monophyletic clades, but are intermixed with each other as well as with the osmotrophic taxa, Astasia and Khawkinea.     

Comparative morphology of the euglenid pellicle. I. Patterns of strips and pores

In anticipation that improved knowledge of euglenid morphology will provide robust apomorphy-based definitions for clades, transmission and scanning electron microscopy were used to reveal novel morphological patterns associated with the euglenid pellicle. In some taxa, the number of pellicle strips around the cell periphery reduces as discrete whorls at the anterior and posterior ends of the cell. The number of whorls at either end varies between selected euglenid taxa but is invariant within a taxon. The pattern of strip reduction associated with these whorls is shown to have at least three evolutionarily linked states: exponential, pseudoexponential, and linear. Two general equations describe these states near the posterior end of euglenid cells. Exponential patterns of strip reduction near the anterior end are described by a third equation. In addition, several euglenid taxa were found to possess conspicuous pellicle pores. These pores are arranged in discrete rows that follow the articulation zones between adjacent strips. The number of strips between rows of pores varies between taxa and displays a series of consecutive character states that differ by a power of two. The patterns of pores may not only have phylogenetical and taxonomical value but may provide morphological markers for following strip maturation during cytoskeletal reproduction.     

A new photosynthetic euglenoid isolated in Poland: Euglenaria clepsydroides sp. nov. (Euglenea)

In this paper, we describe a new photosynthetic euglenoid species, Euglenaria clepsydroides Zakry?, sp. nov., found in Poland. A large population of this species exists in a few, small, eutrophic bodies of water inside the Masurian Landscape Park (covering a part of the Masurian Lake District in Poland). The characteristic and atypical (hourglass-like) cell shape sets it well apart from the other species that have been described up to now. This atypical cell shape has so far been observed only in three species – Lepocinclis constricta, Euglena undulata and Euglena gymnodinioides – whose other morphological characteristics, such as the number and morphology of chloroplasts, the lack of mucocysts, and nuclear SSU rDNA sequence data, exclude the possibility that they could be close relatives of Euglenaria clepsydroides. On the phylogenetic tree, the new species is situated within the Euglenaria clade. While it is a sister group of the clade that includes representatives of Euglenaria anabaena, the two species are clearly morphologically distinct.