To shed light on the disputed molecular phylogeny of Euglenozoa, SSU rDNA sequences of an uncultured Peranema sp. (wild) and cultured Ploeotia edaphica (CCAP 1265/2) were isolated and a database was created containing a great many euglenozoan SSU rDNA sequences. Additionally, new LSU rDNA sequences were isolated from diplonemid Rhynchopus euleeides (ATCC 50226) and Diplonema ambulator (ATCC 50223) as well as of phagotrophic euglenids Entosiphon sulcatum (CCAP 1220/1B), Notosolenus ostium (wild), Peranema trichophorum (CCAP 1260/1B), Petalomonas cantuscygni (CCAP 1259/1), Ploeotia costata (CCAP 1265/1), and primary osmotrophic euglenids Astasia curvata (SAG 1204-5b), Astasia torta (SAG 217.80) and Rhabdomonas costata (SAG 1271-1) by the application of specifically designed primers for primer walking through unknown parts of this understudied gene region.
As a new approach, recently published SSU and LSU rRNA secondary structure data of Saccharomyces cerevisiae (Petrov et al. 2013 and 2014) was utilized as a blueprint for alignment of nucleotide sequences and deduction of secondary structure elements. Several datasets were formed to investigate phylogenetic inferences of SSU rDNA sequences with an equilibrated taxon sampling, separate marine and freshwater lineages and a combined set comprising more than 150 euglenozoan SSU rDNA sequences. Further examinations included two datasets derived from new LSU rDNA sequences as well as a concatenated dataset comprising genetic information of the ribosomal operon for the first time concerning euglenids, diplonemids and kinetoplastids. To address the adherent problem of weakness in statistical support regarding Euglenida found in prior studies, built datasets were additionally examined by phylogenetic network and spectral analyses. These analyses were also used to verify phylogenetic signals of identified monophyla and to test their tree compatibility. Finally, deduced secondary structures were utilized to pinpoint boundaries of coding and spacer regions as a precondition for examination of variable regions of SSU rDNA, SSU and LSU rDNA sequence lengths and corresponding base composition, identity matrices, ITS sequences and their insertion sites in LSU rDNA as well as unique nucleotide substitutions in the search for group specificities among Euglenozoa.
As a result, important findings concerning the phylogeny of major euglenozoan groups have been found, i.e. (1) Euglenida were not monophyletic, for Petalomonadida represented the deepest branch of Euglenozoa and the taxon Euglenida possessed no phylogenetic signal (2) Diplonemida were not the sister group of Kinetoplastida, phylogenetic and secondary structure analyses strongly inferred a relation of Diplonemida with Petalomonadida and Ploeotiida rather than with Kinetoplastida, (3) the existence of a euglenid crown group was confirmed by phylogenetic as well as spectral analyses and according to the eponymous autapomorphy, a helically pellicle pattern, prior taxon denominations were converted into Helicales taxon nov. PAERSCHKE & PREISFELD 2015, (4) the denominations „phagotrophic euglenids‟ and „Heteronematina‟ sensu Adl et al. (2012) describe a polyphyletic assemblage of euglenids and should be discarded.
The present work provides a basis for further examinations of euglenozoan LSU rDNA sequences and thus represents a precursor for future studies concerning the ribosomal operon of Euglenozoa.