Ultrastructure and molecular phylogeny of Stephanopogon minuta: An enigmatic microeukaryote from marine interstitial environments

Although Stephanopogon was described as a putative ciliate more than a century ago, its phylogenetic position within eukaryotes has remained unclear because of an unusual combination of morphological characteristics (e.g. a highly multiflagellated cell with discoidal mitochondrial cristae). Attempts to classify Stephanopogon have included placement with the Ciliophora, the Euglenozoa, the Heterolobosea and the Rhizaria. Most systematists have chosen, instead, to conservatively classify Stephanopogon as incertae sedis within eukaryotes. Despite the obvious utility of molecular phylogenetic data in resolving this issue, DNA sequences from Stephanopogon have yet to be published. Accordingly, we characterized the molecular phylogeny and ultrastructure of Stephanopogon minuta, a species we isolated from marine sediments in southern British Columbia, Canada. Our results showed that S. minuta shares several features with heteroloboseans, such as discoidal mitochondrial cristae, a heterolobosean-specific (17_1 helix) insertion in the small subunit ribosomal RNA gene (SSU rDNA) and the lack of canonical Golgi bodies. Molecular phylogenetic analyses of SSU rDNA demonstrated that S. minuta branches strongly within the Heterolobosea and specifically between two different tetraflagellated lineages, both named ‘Percolomonas cosmopolitus.’ Several ultrastructural features shared by S. minuta and P. cosmopolitus reinforced the molecular phylogenetic data and confirmed that Stephanopogon is a highly divergent multiflagellated heterolobosean that represents an outstanding example of convergent evolution with benthic eukaryovorous ciliates (Alveolata).