Long branch attraction effects and the status of "basal eukaryotes": phylogeny and structural analysis of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis

The three taxa emerging at the base of the eukaryotic ribosomal RNA phylogenetic tree (Diplomonadida, Microspora, and Parabasalia) include a wide array of parasitic species. and some free-living organisms that appear to be derived from a parasitic ancestry. The basal position of these taxa, which lack mitochondria, has recently been questioned. I sequenced most of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis and a secondary structure model was reconstructed for the SSU rRNA. I conducted a RASA matrix analysis to identify, independently from tree reconstruction, putative long branch attraction effects in the data matrix. The results show that each of the basal clades and the euglenozoan clade act, indeed, as long branches and may have been engaged in a process of accelerated rate of evolution. A nucleotide signature analysis was conducted in the conserved regions for positions defining the three great domains of life (Eubacteria, Archea, and Eukaryota). For the three basal taxa, this analysis showed the presence of a significant number of different non-eukaryotic nucleotides. A precise study of the nature and location of these nucleotides led to conclusions supporting the results of the RASA analysis. Altogether, these findings suggest that the basal placement of these taxa in the SSU ribosomal RNA phylogenetic tree is artifactual, and flawed by long branch attraction effects.     

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.     

TRENDS IN THE EVOLUTION OF THE EUGLENID PELLICLE

Abstract Trends in the evolution of the euglenid pellicle were described using phylogenetic methods on 18S rDNA, morphological, and combined data from 25 mostly phototrophic taxa. The tree topology from a total‐evidence analysis formed a template for a synthetic tree that took into account conflicting results derived from the partitioned datasets. Pellicle character states that can only be observed with the assistance of transmission and scanning electron microscopy were phylogenetically mapped onto the synthetic tree to test a set of previously established homology statements (inferences made independently from a cladogram). The results permitted us to more confidently infer the ancestral‐derived polarities of character state transformations and provided a framework for understanding the key cytoskeletal innovations associated with the evolution of phototrophic euglenids. We specifically addressed the character evolution of (1) the maximum number of pellicle strips around the cell periphery; (2) the patterns of terminating strips near the cell posterior end; (3) the substructural morphology of pellicle strips; (4) the morphology of the cell posterior tip; and (5) patterns of pellicle pores on the cell surface.     

FEEDING IN PERANEMA TRICHOPHORUM REVISITED (EUGLENOPHYTA)1

Feeding in Peranema trichophorum (Ehrenberg 1838) Stein 1878 was first observed over a century ago, yet there is still contention over how the feeding apparatus is used in feeding. Using video microscopy and scanning microscopy, this study documents two types of feeding. Peranema may engulf prey cells whole. Immotile cells are preferred, but moving cells occasionally are engulfed. Details of the early stages of engulfment are presented using scanning electron microscopy. A second method of feeding begins with the attachment of Peranema to a prey cell. The rods of the feeding apparatus then are repeatedly scraped over the surface of the prey until a tear occurs in the cell. The feeding apparatus is inserted into the opening and the internal cell contents are sucked out. The anterior flagellum is inserted into the prey to help remove the cell's contents.     

Phylogeny of phagotrophic euglenids (Euglenozoa) as inferred from hsp90 gene sequences

Molecular phylogenies of euglenids are usually based on ribosomal RNA genes that do not resolve the branching order among the deeper lineages. We addressed deep euglenid phylogeny using the cytosolic form of the heat-shock protein 90 gene (hsp90), which has already been employed with some success in other groups of euglenozoans and eukaryotes in general. Hsp90 sequences were generated from three taxa of euglenids representing different degrees of ultrastructural complexity, namely Petalomonas cantuscygni and wild isolates of Entosiphon sulcatum, and Peranema trichophorum. The hsp90 gene sequence of P. trichophorum contained three short introns (ranging from 27 to 31 bp), two of which had non-canonical borders GG-GG and GG-TG and two 10-bp inverted repeats, suggesting a structure similar to that of the non-canonical introns described in Euglena gracilis. Phylogenetic analyses confirmed a closer relationship between kinetoplastids and diplonemids than to euglenids, and supported previous views regarding the branching order among primarily bacteriovorous, primarily eukaryovorous, and photosynthetic euglenids. The position of P. cantuscygni within Euglenozoa, as well as the relative support for the nodes including it were strongly dependent on outgroup selection. The results were most consistent when the jakobid Reclinomonas americana was used as the outgroup. The most robust phylogenies place P. cantuscygni as the most basal branch within the euglenid clade. However, the presence of a kinetoplast-like mitochondrial inclusion in P. cantuscygni deviates from the currently accepted apomorphy-based definition of the kinetoplastid clade and highlights the necessity of detailed studies addressing the molecular nature of the euglenid and diplonemid mitochondrial genome.