Euglenozoan kleptoplasty illuminates the early evolution of photoendosymbiosis.

Kleptoplasts (kP) are distinct among photosynthetic organelles in eukaryotes (i.e., plastids) because they are routinely sequestered from prey algal cells and function only temporarily in the new host cell. Therefore, the hosts of kleptoplasts benefit from photosynthesis without constitutive photoendosymbiosis. Here, we report that the euglenozoan Rapaza viridis has only kleptoplasts derived from a specific strain of green alga, Tetraselmis sp., but no canonical plastids like those found in its sister group, the Euglenophyceae. R. viridis showed a dynamic change in the accumulation of cytosolic polysaccharides in response to light-dark cycles, and 13C isotopic labeling of ambient bicarbonate demonstrated that these polysaccharides originate in situ via photosynthesis; these data indicate that the kleptoplasts of R. viridis are functionally active. We also identified 276 sequences encoding putative plastid-targeting proteins and 35 sequences of presumed kleptoplast transporters in the transcriptome of R. viridis. These genes originated in a wide range of algae other than Tetraselmis sp., the source of the kleptoplasts, suggesting a long history of repeated horizontal gene transfer events from different algal prey cells. Many of the kleptoplast proteins, as well as the protein-targeting system, in R. viridis were shared with members of the Euglenophyceae, providing evidence that the early evolutionary stages in the green alga-derived secondary plastids of euglenophytes also involved kleptoplasty.

Refurbishing the marine parasitoid order Pirsoniales with newly (re)described marine and freshwater free-living predators.

Pirsoniales is a stramenopile order composed of marine parasitoids of diatoms with unique life cycle. Until recently, a single genus, Pirsonia, uniting six species, was known. The recent identification of new free-living eukaryotrophic Pirsoniales Pirsonia chemainus, Feodosia pseudopoda, and Koktebelia satura changed our understanding of this group as exclusively parasitic. However, their cell ultrastructure and feeding preferences were not fully studied due to the death of the cultures. In this study, we re-isolated some of these Pirsoniales and established six new strains exhibiting predatory behavior, including a first freshwater representative. This allowed us to describe five new genera and species, as well as to emend the diagnosis of the order Pirsoniales. The 18S rRNA gene phylogenetic analysis revealed the position of new strains within Pirsoniales and their relationships with parasitoid relatives and environmental sequence lineages. Feeding experiments on novel Pirsoniales strains using diverse algal prey showed that they were not able to form trophosomes and auxosomes. The ability of cell aggregation in Pirsoniales was observed for the first time. One of the studied strains contained intracellular gammaproteobacteria distantly related to Coxiella. Ultrastructural analyses revealed a more complex cytoskeleton structure in Pirsoniales than previously thought and supported the monophyly of Bigyromonadea and Pseudofungi.

Expanding the molecular and morphological diversity of Apusomonadida, a deep-branching group of gliding bacterivorous protists.

Apusomonads are cosmopolitan bacterivorous biflagellate protists usually gliding on freshwater and marine sediment or wet soils. These nanoflagellates form a sister lineage to opisthokonts and may have retained ancestral features helpful to understanding the early evolution of this large supergroup. Although molecular environmental analyses indicate that apusomonads are genetically diverse, few species have been described. Here, we morphologically characterize 11 new apusomonad strains. Based on molecular phylogenetic analyses of the rRNA gene operon, we describe four new strains of the known species Multimonas media, Podomonas capensis, Apusomonas proboscidea, and Apusomonas australiensis, and rename Thecamonas oxoniensis as Mylnikovia oxoniensis n. gen., n. comb. Additionally, we describe four new genera and six new species: Catacumbia lutetiensis n. gen. n. sp., Cavaliersmithia chaoae n. gen. n. sp., Singekia montserratensis n. gen. n. sp., Singekia franciliensis n. gen. n. sp., Karpovia croatica n. gen. n. sp., and Chelonemonas dolani n. sp. Our comparative analysis suggests that apusomonad ancestor was a fusiform biflagellate with a dorsal pellicle, a plastic ventral surface, and a sleeve covering the anterior flagellum, that thrived in marine, possibly oxygen-poor, environments. It likely had a complex cell cycle with dormant and multiple fission stages, and sex. Our results extend known apusomonad diversity, allow updating their taxonomy, and provide elements to understand early eukaryotic evolution.

Molecular and morphological characterization of four new ancyromonad genera and proposal for an updated taxonomy of the Ancyromonadida.

Ancyromonads are small biflagellated protists with a bean-shaped morphology. They are cosmopolitan in marine, freshwater, and soil environments, where they attach to surfaces while feeding on bacteria. These poorly known grazers stand out by their uncertain phylogenetic position in the tree of eukaryotes, forming a deep-branching "orphan" lineage that is considered key to a better understanding of the early evolution of eukaryotes. Despite their ecological and evolutionary interest, only limited knowledge exists about their true diversity. Here, we aimed to characterize ancyromonads better by integrating environmental surveys with behavioral observation and description of cell morphology, for which sample isolation and culturing are indispensable. We studied 18 ancyromonad strains, including 14 new isolates and seven new species. We described three new and genetically divergent genera: Caraotamonas, Nyramonas, and Olneymonas, together encompassing four species. The remaining three new species belong to the already-known genera Fabomonas and Ancyromonas. We also raised Striomonas, formerly a subgenus of Nutomonas, to full genus status, on morphological and phylogenetic grounds. We studied the morphology of diverse ancyromonads under light and electron microscopy and carried out molecular phylogenetic analyses, also including 18S rRNA gene sequences from several environmental surveys. Based on these analyses, we have updated the taxonomy of Ancyromonadida.

A new lineage of non-photosynthetic green algae with extreme organellar genomes.

BACKGROUND: The plastid genomes of the green algal order Chlamydomonadales tend to expand their non-coding regions, but this phenomenon is poorly understood. Here we shed new light on organellar genome evolution in Chlamydomonadales by studying a previously unknown non-photosynthetic lineage. We established cultures of two new Polytoma-like flagellates, defined their basic characteristics and phylogenetic position, and obtained complete organellar genome sequences and a transcriptome assembly for one of them. RESULTS: We discovered a novel deeply diverged chlamydomonadalean lineage that has no close photosynthetic relatives and represents an independent case of photosynthesis loss. To accommodate these organisms, we establish the new genus Leontynka, with two species (L. pallida and L. elongata) distinguishable through both their morphological and molecular characteristics. Notable features of the colourless plastid of L. pallida deduced from the plastid genome (plastome) sequence and transcriptome assembly include the retention of ATP synthase, thylakoid-associated proteins, the carotenoid biosynthesis pathway, and a plastoquinone-based electron transport chain, the latter two modules having an obvious functional link to the eyespot present in Leontynka. Most strikingly, the ~362 kbp plastome of L. pallida is by far the largest among the non-photosynthetic eukaryotes investigated to date due to an extreme proliferation of sequence repeats. These repeats are also present in coding sequences, with one repeat type found in the exons of 11 out of 34 protein-coding genes, with up to 36 copies per gene, thus affecting the encoded proteins. The mitochondrial genome of L. pallida is likewise exceptionally large, with its >104 kbp surpassed only by the mitogenome of Haematococcus lacustris among all members of Chlamydomonadales hitherto studied. It is also bloated with repeats, though entirely different from those in the L. pallida plastome, which contrasts with the situation in H. lacustris where both the organellar genomes have accumulated related repeats. Furthermore, the L. pallida mitogenome exhibits an extremely high GC content in both coding and non-coding regions and, strikingly, a high number of predicted G-quadruplexes. CONCLUSIONS: With its unprecedented combination of plastid and mitochondrial genome characteristics, Leontynka pushes the frontiers of organellar genome diversity and is an interesting model for studying organellar genome evolution.

Ancient Adaptive Lateral Gene Transfers in the Symbiotic Opalina-Blastocystis Stramenopile Lineage.

Lateral gene transfer is a very common process in bacterial and archaeal evolution, playing an important role in the adaptation to new environments. In eukaryotes, its role and frequency remain highly debated, although recent research supports that gene transfer from bacteria to diverse eukaryotes may be much more common than previously appreciated. However, most of this research focused on animals and the true phylogenetic and functional impact of bacterial genes in less-studied microbial eukaryotic groups remains largely unknown. Here, we have analyzed transcriptome data from the deep-branching stramenopile Opalinidae, common members of frog gut microbiomes, and distantly related to the well-known genus Blastocystis. Phylogenetic analyses suggest the early acquisition of several bacterial genes in a common ancestor of both lineages. Those lateral gene transfers most likely facilitated the adaptation of the free-living ancestor of the Opalinidae-Blastocystis symbiotic group to new niches in the oxygen-depleted animal gut environment.

Ultrastructure and Molecular Phylogeny of Iotanema spirale gen. nov. et sp. nov., a New Lineage of Endobiotic Fornicata with Strikingly Simplified Ultrastructure.

Fornicata (Metamonada) is a group of Excavata living in low-oxygen environments and lacking conventional mitochondria. It includes free-living Carpediemonas-like organisms from marine habitats and predominantly parasitic/commensal retortamonads and diplomonads. Current modest knowledge of biodiversity of Fornicata limits our ability to draw a complete picture of the evolutionary history in this group. Here, we report the discovery of a novel fornicate, Iotanema spirale gen. nov. et sp. nov., obtained from fresh feces of the gecko Phelsuma madagascariensis. Our phylogenetic analyses of the small subunit ribosomal RNA gene demonstrate that I. spirale is closely related to the free-living, marine strain PCS and the Carpediemonas-like organism Hicanonectes teleskopos within Fornicata. Iotanema spirale exhibits several features uncommon to fornicates, such as a single flagellum, a highly reduced cytoskeletal system, and the lack of the excavate ventral groove, but shares these characters with the poorly known genus Caviomonas. Therefore, I. spirale is accommodated within the family Caviomonadidae, which represents the third known endobiotic lineage of Fornicata. This study improves our understanding of character evolution within Fornicata when placed within the molecular phylogenetic context.

Morphological Identities of Two Different Marine Stramenopile Environmental Sequence Clades: Bicosoeca kenaiensis (Hilliard, 1971) and Cantina marsupialis (Larsen and Patterson, 1990) gen. nov., comb. nov.

Although environmental DNA surveys improve our understanding of biodiversity, interpretation of unidentified lineages is limited by the absence of associated morphological traits and living cultures. Unidentified lineages of marine stramenopiles are called "MAST clades". Twenty-five MAST clades have been recognized: MAST-1 through MAST-25; seven of these have been subsequently discarded because the sequences representing those clades were found to either (1) be chimeric or (2) affiliate within previously described taxonomic groups. Eighteen MAST clades remain without a cellular identity. Moreover, the discarded "MAST-13" has been used in different studies to refer to two different environmental sequence clades. After establishing four cultures representing two different species of heterotrophic stramenopiles and then characterizing their morphology and molecular phylogenetic positions, we determined that the two different species represented the two different MAST-13 clades: (1) a lorica-bearing Bicosoeca kenaiensis and (2) a microaerophilic flagellate previously named "Cafeteria marsupialis". Both species were previously described with only light microscopy; no cultures, ultrastructural data or DNA sequences were available from these species prior to this study. The molecular phylogenetic position of three different "C. marsupialis" isolates was not closely related to the type species of Cafeteria; therefore, we established a new genus for these isolates, Cantina gen. nov.

Evolution of microtubule organizing centers across the tree of eukaryotes.

The 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.

The evolution of paralogous enzymes MAT and MATX within the Euglenida and beyond.

BACKGROUND: Methionine adenosyltransferase (MAT) is a ubiquitous essential enzyme that, in eukaryotes, occurs in two relatively divergent paralogues: MAT and MATX. MATX has a punctate distribution across the tree of eukaryotes and, except for a few cases, is mutually exclusive with MAT. This phylogenetic pattern could have arisen by either differential loss of old paralogues or the spread of one of these paralogues by horizontal gene transfer. Our aim was to map the distribution of MAT/MATX genes within the Euglenida in order to more comprehensively characterize the evolutionary history of MATX. RESULTS: We generated 26 new sequences from 23 different lineages of euglenids and one prasinophyte alga Pyramimonas parkeae. MATX was present only in photoautotrophic euglenids. The mixotroph Rapaza viridis and the prasinophyte alga Pyramimonas parkeae, which harbors chloroplasts that are most closely related to the chloroplasts in photoautotrophic euglenids, both possessed only the MAT paralogue. We found both the MAT and MATX paralogues in two photoautotrophic species (Phacus orbicularis and Monomorphina pyrum). The significant conflict between eukaryotic phylogenies inferred from MATX and SSU rDNA data represents strong evidence that MATX paralogues have undergone horizontal gene transfer across the tree of eukaryotes. CONCLUSIONS: Our results suggest that MATX entered the euglenid lineage in a single horizontal gene transfer event that took place after the secondary endosymbiotic origin of the euglenid chloroplast. The origin of the MATX paralogue is unclear, and it cannot be excluded that it arose by a gene duplication event before the most recent common ancestor of eukaryotes.

A resurgence in field research is essential to better understand the diversity, ecology, and evolution of microbial eukaryotes.

The discovery and characterization of protist communities from diverse environments are crucial for understanding the overall evolutionary history of life on earth. However, major questions about the diversity, ecology, and evolutionary history of protists remain unanswered, notably because data obtained from natural protist communities, especially of heterotrophic species, remain limited. In this review, we discuss the challenges associated with "field protistology", defined here as the exploration, characterization, and interpretation of microbial eukaryotic diversity within the context of natural environments or field experiments, and provide suggestions to help fill this important gap in knowledge. We also argue that increased efforts in field studies that combine molecular and microscopical methods offer the most promising path toward (1) the discovery of new lineages that expand the tree of eukaryotes; (2) the recognition of novel evolutionary patterns and processes; (3) the untangling of ecological interactions and functions, and their roles in larger ecosystem processes; and (4) the evaluation of protist adaptations to a changing climate.

Parallel re-modeling of EF-1α function: divergent EF-1α genes co-occur with EFL genes in diverse distantly related eukaryotes.

BACKGROUND: Elongation factor-1α (EF-1α) and elongation factor-like (EFL) proteins are functionally homologous to one another, and are core components of the eukaryotic translation machinery. The patchy distribution of the two elongation factor types across global eukaryotic phylogeny is suggestive of a 'differential loss' hypothesis that assumes that EF-1α and EFL were present in the most recent common ancestor of eukaryotes followed by independent differential losses of one of the two factors in the descendant lineages. To date, however, just one diatom and one fungus have been found to have both EF-1α and EFL (dual-EF-containing species). RESULTS: In this study, we characterized 35 new EF-1α/EFL sequences from phylogenetically diverse eukaryotes. In so doing we identified 11 previously unreported dual-EF-containing species from diverse eukaryote groups including the Stramenopiles, Apusomonadida, Goniomonadida, and Fungi. Phylogenetic analyses suggested vertical inheritance of both genes in each of the dual-EF lineages. In the dual-EF-containing species we identified, the EF-1α genes appeared to be highly divergent in sequence and suppressed at the transcriptional level compared to the co-occurring EFL genes. CONCLUSIONS: According to the known EF-1α/EFL distribution, the differential loss process should have occurred independently in diverse eukaryotic lineages, and more dual-EF-containing species remain unidentified. We predict that dual-EF-containing species retain the divergent EF-1α homologues only for a sub-set of the original functions. As the dual-EF-containing species are distantly related to each other, we propose that independent re-modelling of EF-1α function took place in multiple branches in the tree of eukaryotes.

Ultrastructure and molecular phylogenetic position of Heteronema scaphurum: a eukaryovorous euglenid with a cytoproct.

Euglenids comprise a distinct clade of flagellates with diverse modes of nutrition, including phagotrophy, osmotrophy and phototrophy. Much of the previous research on euglenids has focused on phototrophic species because of their ecological abundance and significance as indicators for the health of aquatic ecosystems. Although largely understudied, phagotrophic species probably represent the majority of euglenid diversity. Phagotrophic euglenids tend to be either bacterivorous or eukaryovorous and use an elaborate feeding apparatus for capturing prey cells. We characterized the ultrastructure and molecular phylogenetic position of Heteronema scaphurum, a eukaryovorous euglenid collected in freshwater. This species was equipped with a distinct cytoproct through which waste products were eliminated in the form of faecal pellets; a cytoproct has not been reported in any other member of the Euglenida. Heteronema scaphurum also had a novel predatory mode of feeding. The euglenid ensnared and corralled several green algal prey cells (i.e. Chlamydomonas) with hook-like flagella covered in mucous before engulfing the bundle of prey cells whole. Molecular phylogenetic analyses inferred from small subunit rDNA sequences placed this species with other eukaryovorous euglenids, which was consistent with ultrastructural features associated with the feeding apparatus, flagellar apparatus, extrusomes, and pellicle.

Phylogenetic and morphological diversity of free-living diplomonads.

Diplomonadida is a lineage of anaerobic protists belonging to Fornicata, Metamonada. Most diplomonads are endobiotic or parasitic, such as Giardia intestinalis, which is a famous human pathogen, but several free-living species exist as well. Although it has been proposed that the free-living diplomonads are descendants of endobiotic organisms and thus interesting from the evolutionary point of view, they have been largely neglected. We obtained 58 cultures of free-living diplomonads belonging to four genera (Hexamita, Trepomonas, Gyromonas, and Trimitus) and six strains of endobiotic diplomonads and analyzed their SSU rRNA gene sequences. We also studied light-microscopic morphology of selected strains and the ultrastructure of Trepomonas rotans for the first time. Our phylogenetic analysis showed that the genus Hexamita, and, possibly, also the genus Trepomonas, are polyphyletic. Trepomonas rotans, which may represent a novel genus, is unique among Diplomonadida by having the cell covered in scales. Our results suggest that the evolution of the endobiotic life style and cell organization in diplomonads is more complicated than previously thought.

Morphostasis in a novel eukaryote illuminates the evolutionary transition from phagotrophy to phototrophy: description of Rapaza viridis n. gen. et sp. (Euglenozoa, Euglenida).

BACKGROUND: Morphostasis 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. RESULTS: We 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. CONCLUSIONS: The 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.

Free-living Trichomonads are Unexpectedly Diverse.

The vast majority of the more than 450 described species of Parabasalia are intestinal symbionts or parasites of animals. This endobiotic life-history is presumably ancestral although the root of Parabasalia still needs to be robustly established. The half-dozen putatively free-living species thus far described are likely independently derived from endobiotic ancestors and represent the most neglected ecological group of parabasalids. Thus, we isolated and cultivated 45 free-living strains of Parabasalia obtained from a wide variety of anoxic sediments to conduct detailed morphological and SSU rRNA gene phylogenetic analyses. Sixteen species of trichomonads were recovered. Among them, we described seven new species, three new genera, two new families, and one new order. Most of the newly described species were more or less closely related to members of already described genera. However, we uncovered a new deep-branching lineage without affinity to any currently known group of Parabasalia. The newly discovered free-living parabasalids will be key taxa in comparative analyses aimed at rooting the entire lineage and deciphering the evolutionary innovations involved in transitioning between endobiotic and free-living habitats.

Tsukubamonas globosa n. gen., n. sp., a novel excavate flagellate possibly holding a key for the early evolution in "Discoba".

We report the ultrastructure and phylogenetic position of a free-living heterotrophic flagellate, Tsukubamonas globosa n. gen., n. sp. This flagellate was isolated from a pond in the University of Tsukuba, Japan. Under light microscopy, the spherical vegetative cells were naked and highly vacuolated, and always swam with rotating motion. Electron microscopic observations revealed that T. globosa possessed a ventral feeding groove, which is one of the hallmark characteristics of the supergroup Excavata. The position of T. globosa was unresolved in the small subunit ribosomal RNA phylogeny. On the other hand, a multigene phylogeny using α-tubulin, ß-tubulin, actin, heat shock protein 90, and translation elongation factor 2 robustly united T. globosa with members of the "Discoba" clade of Excavata, composed of jakobids, euglenozoans, and heteroloboseans, although the precise position of T. globosa in this clade remained unresolved. Our detailed morphological comparisons elucidated that T. globosa possessed a novel set of morphological features, and could not be classified into any taxa in the Discoba clade. Instead we classified T. globosa into Tsukubamonadidae n. fam. under Tsukubamonadida n. ord.

A wide diversity of previously undetected free-living relatives of diplomonads isolated from marine/saline habitats.

Over the last 15 years classical culturing and environmental PCR techniques have revealed a modest number of genuinely new major lineages of protists; however, some new groups have greatly influenced our understanding of eukaryote evolution. We used culturing techniques to examine the diversity of free-living protists that are relatives of diplomonads and retortamonads, a group of evolutionary and parasitological importance. Until recently, a single organism, Carpediemonas membranifera, was the only representative of this region of the tree. We report 18 new isolates of Carpediemonas-like organisms (CLOs) from anoxic marine sediments. Only one is a previously cultured species. Eleven isolates are conspecific and were classified within a new genus, Kipferlia n. gen. The remaining isolates include representatives of three other lineages that likely represent additional undescribed genera (at least). Small-subunit ribosomal RNA gene phylogenies show that CLOs form a cloud of six major clades basal to the diplomonad-retortamonad grouping (i.e. each of the six CLO clades is potentially as phylogenetically distinct as diplomonads and retortamonads). CLOs will be valuable for tracing the evolution of diplomonad cellular features, for example, their extremely reduced mitochondrial organelles. It is striking that the majority of CLO diversity was undetected by previous light microscopy surveys and environmental PCR studies, even though they inhabit a commonly sampled environment. There is no reason to assume this is a unique situation - it is likely that undersampling at the level of major lineages is still widespread for protists.

Ultrastructure and molecular phylogenetic position of a novel euglenozoan with extrusive episymbiotic bacteria: Bihospites bacati n. gen. et sp. (Symbiontida).

BACKGROUND: Poorly understood but highly diverse microbial communities exist within anoxic and oxygen-depleted marine sediments. These communities often harbour single-celled eukaryotes that form symbiotic associations with different prokaryotes. During low tides in South-western British Columbia, Canada, vast areas of marine sand become exposed, forming tidal pools. Oxygen-depleted sediments within these pools are distinctively black at only 2-3 cm depth; these layers contain a rich variety of microorganisms, many of which are undescribed. We discovered and characterized a novel (uncultivated) lineage of heterotrophic euglenozoan within these environments using light microscopy, scanning and transmission electron microscopy, serial sectioning and ultrastructural reconstruction, and molecular phylogenetic analyses of small subunit rDNA sequences. RESULTS: Bihospites bacati n. gen. et sp. is a biflagellated microbial eukaryote that lives within low-oxygen intertidal sands and dies within a few hours of exposure to atmospheric oxygen. The cells are enveloped by two different prokaryotic episymbionts: (1) rod-shaped bacteria and (2) longitudinal strings of spherical bacteria, capable of ejecting an internal, tightly wound thread. Ultrastructural data showed that B. bacati possesses all of the euglenozoan synapomorphies. Moreover, phylogenetic analyses of SSU rDNA sequences demonstrated that B. bacati groups strongly with the Symbiontida: a newly established subclade within the Euglenozoa that includes Calkinsia aureus and other unidentified organisms living in low-oxygen sediments. B. bacati also possessed novel features, such as a compact C-shaped rod apparatus encircling the nucleus, a cytostomal funnel and a distinctive cell surface organization reminiscent of the pellicle strips in phagotrophic euglenids. CONCLUSIONS: We characterized the ultrastructure and molecular phylogenetic position of B. bacati n. gen. et sp. Molecular phylogenetic analyses demonstrated that this species belongs to the Euglenozoa and currently branches as the earliest diverging member of the Symbiontida. This is concordant with ultrastructural features of B. bacati that are intermediate between C. aureus and phagotrophic euglenids, indicating that the most recent ancestor of the Symbiontida descended from phagotrophic euglenids. Additionally, the extrusive episymbionts in B. bacati are strikingly similar to so-called "epixenosomes", prokaryotes previously described in a ciliate species and identified as members of the Verrucomicrobia. These parallel symbioses increase the comparative context for understanding the origin(s) of extrusive organelles in eukaryotes and underscores how little we know about the symbiotic communities of marine benthic environments.

Cryptic diversity of free-living parabasalids, Pseudotrichomonas keilini and Lacusteria cypriaca n. g., n. sp., as inferred from small subunit rDNA sequences.

Ultrastructural and molecular phylogenetic evidence indicate that the Parabasalia consists of seven main subgroups: the Trichomonadida, Honigbergiellida, Hypotrichomonadida, Tritrichomonadida, Cristamonadida, Spirotrichonymphida, and Trichonymphida. Only five species of free-living parabasalids are known: Monotrichomonas carabina, Ditrichomonas honigbergii, Honigbergiella sp., Tetratrichomonas undula, and Pseudotrichomonas keilini. Phylogenetic analyses show that free-living species do not form a clade and instead branch in several different positions within the context of their parasitic relatives. Because the diversity of free-living parabasalids is poorly understood, the systematics of these lineages is in a significant state of disarray. In order to better understand the phylogenetic distribution of free-living parabasalids, we sequenced the small subunit rDNA from three different strains reminiscent of P. keilini; the strains were isolated from different geographical locations: (1) mangrove sediments in Japan and (2) sediments in Cyprus. These data demonstrated that the free-living parabasalids P. keilini and Lacusteria cypriaca n. g., n. sp., form a paraphyletic assemblage near the origin of a clade consisting mostly of parasitic trichomonadids (e.g. Trichomonas vaginalis). This paraphyletic distribution of similar morphotypes indicates that free-living trichomonadids represent a compelling example of morphostasis that provides insight into the suite of features present in the most recent free-living ancestor of their parasitic relatives.