Recent culture-independent surveys of eukaryotic small-subunit ribosomal DNA (SSU rDNA) from many environments have unveiled unexpectedly high diversity of microbial eukaryotes (microeukaryotes) at various taxonomic levels. However, such surveys were most probably biased by various technical difficulties, resulting in underestimation of microeukaryotic diversity. In the present study on oxygen-depleted sediment from a deep-sea methane cold seep of Sagami Bay, Japan, we surveyed the diversity of eukaryotic rDNA in raw sediment samples and in two enrichment cultures. More than half of all clones recovered from the raw sediment samples were of the basidiomycetous fungus Cryptococcus curvatus. Among other clones, phylotypes of eukaryotic parasites, such as Apicomplexa, Ichthyosporea, and Phytomyxea, were identified. On the other hand, we observed a marked difference in phylotype composition in the enrichment samples. Several phylotypes belonging to heterotrophic stramenopiles were frequently found in one enrichment culture, while a phylotype of Excavata previously detected at a deep-sea hydrothermal vent dominated the other. We successfully established a clonal culture of this excavate flagellate. Since these phylotypes were not identified in the raw sediment samples, the approach incorporating a cultivation step successfully found at least a fraction of the "hidden" microeukaryotic diversity in the environment examined.

BackgroundMorphostasis of traits in different species is necessary for reconstructing the evolutionary history of complex characters. Studies that place these species into a molecular phylogenetic context test hypotheses about the transitional stages that link divergent character states. For instance, the transition from a phagotrophic mode of nutrition to a phototrophic lifestyle has occurred several times independently across the tree of eukaryotes; one of these events took place within the Euglenida, a large group of flagellates with diverse modes of nutrition. Phototrophic euglenids form a clade that is nested within lineages of phagotrophic euglenids and that originated through a secondary endosymbiosis with green algae. Although it is clear that phototrophic euglenids evolved from phagotrophic ancestors, the morphological disparity between species representing these different nutritional modes remains substantial.ResultsWe cultivated a novel marine euglenid, Rapaza viridis n. gen. et sp. ("green grasper"), and a green alga, Tetraselmis sp., from the same environment. Cells of R. viridis were comprehensively characterized with light microscopy, SEM, TEM, and molecular phylogenetic analysis of small subunit rDNA sequences. Ultrastructural and behavioral observations demonstrated that this isolate habitually consumes a specific strain of Tetraselmis prey cells and possesses a functional chloroplast that is homologous with other phototrophic euglenids. A novel feeding apparatus consisting of a reduced rod of microtubules facilitated this first and only example of mixotrophy among euglenids. R. viridis also possessed a robust photoreception apparatus, two flagella of unequal length, euglenoid movement, and a pellicle consisting of 16 strips and one (square-shaped) whorl of posterior strip reduction. The molecular phylogenetic data demonstrated that R. viridis branches as the nearest sister lineage to phototrophic euglenids.ConclusionsThe unusual combination of features in R. viridis combined with its molecular phylogenetic position completely conforms to the expected transitional stage that occurred during the early evolution of phototrophic euglenids from phagotrophic ancestors. The marine mixotrophic mode of nutrition, the preference for green algal prey cells, the structure of the feeding apparatus, and the organization of the pellicle are outstanding examples of morphostasis that clarify pivotal stages in the evolutionary history of this diverse group of microbial eukaryotes.

SUMMARYThe architecture of eukaryotic cells is underpinned by complex arrrays of microtubules that stem from an organizing center, referred to as the MTOC. With few exceptions, MTOCs consist of two basal bodies that anchor flagellar axonemes and different configurations of microtubular roots. Variations in the structure of this cytoskeletal system, also referred to as the 'flagellar apparatus', reflect phylogenetic relationships and provide compelling evidence for inferring the overall tree of eukaryotes. However, reconstructions and subsequent comparisons of the flagellar apparatus are challenging, because these studies require sophisticated microscopy, spatial reasoning and detailed terminology. In an attempt to understand the unifying features of MTOCs and broad patterns of cytoskeletal homology across the tree of eukaryotes, we present a comprehensive overview of the eukaryotic flagellar apparatus within a modern molecular phylogenetic context. Specifically, we used the known cytoskeletal diversity within major groups of eukaryotes to infer the unifying features (ancestral states) for the flagellar apparatus in the Plantae, Opisthokonta, Amoebozoa, Stramenopiles, Alveolata, Rhizaria, Excavata, Cryptophyta, Haptophyta, Apusozoa, Breviata and Collodictyonidae. We then mapped these data onto the tree of eukaryotes in order to trace broad patterns of trait changes during the evolutionary history of the flagellar apparatus. This synthesis suggests that: (i) the most recent ancestor of all eukaryotes already had a complex flagellar apparatus, (ii) homologous traits associated with the flagellar apparatus have a punctate distribution across the tree of eukaryotes, and (iii) streamlining (trait losses) of the ancestral flagellar apparatus occurred several times independently in eukaryotes.

BackgroundThe Euglenozoa is a large group of eukaryotic flagellates with diverse modes of nutrition. The group consists of three main subclades – euglenids, kinetoplastids and diplonemids – that have been confirmed with both molecular phylogenetic analyses and a combination of shared ultrastructural characteristics. Several poorly understood lineages of putative euglenozoans live in anoxic environments, such as Calkinsia aureus, and have yet to be characterized at the molecular and ultrastructural levels. Improved understanding of these lineages is expected to shed considerable light onto the ultrastructure of prokaryote-eukaryote symbioses and the associated cellular innovations found within the Euglenozoa and beyond.ResultsWe collected Calkinsia aureus from core samples taken from the low-oxygen seafloor of the Santa Barbara Basin (580 – 592 m depth), California. These biflagellates were distinctively orange in color and covered with a dense array of elongated epibiotic bacteria. Serial TEM sections through individually prepared cells demonstrated that C. aureus shares derived ultrastructural features with other members of the Euglenozoa (e.g. the same paraxonemal rods, microtubular root system and extrusomes). However, C. aureus also possessed several novel ultrastructural systems, such as modified mitochondria (i.e. hydrogenosome-like), an "extrusomal pocket", a highly organized extracellular matrix beneath epibiotic bacteria and a complex flagellar transition zone. Molecular phylogenies inferred from SSU rDNA sequences demonstrated that C. aureus grouped strongly within the Euglenozoa and with several environmental sequences taken from low-oxygen sediments in various locations around the world.ConclusionCalkinsia aureus possesses all of the synapomorphies for the Euglenozoa, but lacks traits that are specific to any of the three previously recognized euglenozoan subgroups. Molecular phylogenetic analyses of C. aureus demonstrate that this lineage is a member of a novel euglenozoan subclade consisting of uncharacterized cells living in low-oxygen environments. Our ultrastructural description of C. aureus establishes the cellular identity of a fourth group of euglenozoans, referred to as the "Symbiontida".