BACKGROUND
the euglenozoa is a diverse group of single-celled eukaryotes consisting of three main subgroups: euglenids, kinetoplastids and diplonemids.
euglenids are united by the presence of a distinctive pellicle, a superficial system formed by four major components: the plasma membrane, a pattern of repeating proteinaceous strips that run along the length of the cell, subtending microtubules and tubular cisternae of endoplasmic reticulum  <cit> .
the group is widely known for its photosynthetic members , but the majority of the species are heterotrophic .
photosynthetic euglenids evolved from phagotrophic ancestors with a complex feeding apparatus and a large number of pellicle strips that facilitate a characteristic peristaltic cell movement called "euglenoid movement".
this combination of characters allows phagotrophic euglenids to engulf large prey cells, such as eukaryotic algae, which eventually led to the acquisition of chloroplasts via secondary endosymbiosis  <cit> .
euglenids are closely related to kinetoplastids and diplonemids.
kinetoplastids  are united by the presence of a mitochondrial inclusion of distinctively arranged dna molecules, called a kinetoplast or kdna  <cit> .
kinetoplastids and euglenids share several morphological features, such as flagella with hairs and heteromorphic paraxial rods  and mitochondria with paddle-shaped  cristae  <cit> .
diplonemids, on the other hand, possess a large mitochondrion with flattened cristae and apparently lack flagellar hairs  <cit> .
the monophyly of the euglenozoa has been established on the basis of both molecular phylogenetic analyses and the following morphological synapomorphies: a tripartite flagellar root system, presence of heteromorphic paraxial rods and tubular extrusomes.
environmental sequencing of oxygen depleted sediments around the world has shown that these habitats harbour a vast and unknown diversity of microbial lineages  <cit> .
phylogenetic analyses of these data have helped demonstrate the existence of several novel lineages associated with many different eukaryotic supergroups.
although these types of analyses are very effective in revealing the actual diversity of microbes living in a particular environment, these approaches also generate vast amounts of "orphan" data that cannot be linked directly to organisms known from comparative morphology.
nonetheless, some of the environmental sequences recovered from oxygen depleted environments cluster with euglenozoans in phylogenetic analyses but with no clear position within the group  <cit> .
other studies have explored and characterized the microbial diversity in oxygen-depleted environments using microscopical approaches  <cit> .
this research has shown that a reoccurring feature of euglenozoans living in low oxygen environments is the presence of episymbiotic bacteria on the cell surface.
here, we report on a highly unusual  euglenozoan isolated from oxygen depleted marine sediments that is covered with two very different morphotypes of episymbionts.
we characterized this lineage with light microscopy, sem, comprehensive tem, and molecular phylogenetic analyses of ssu rdna sequences.
our data demonstrate that this organism is the earliest diverging member of the symbiontida, which is an emerging subclade of euglenozoans composed of anaerobic and microaerophilic flagellates with a superficial layer of mitochondrion-derived organelles that associates closely with a uniform layer of episymbiotic bacteria  <cit> .
moreover, the comparative ultrastructural data from this novel lineage sheds considerable light onto the phylogenetic position of the symbiontida, as a whole, within the euglenozoa.
RESULTS
general morphology
the cells of bihospites bacati n. gen. et sp.
were elongated with a somewhat rounded posterior end and were 40- <dig> μm long and 15- <dig> μm wide .
the cells contained a brownish  body near the posterior end of the cell and a variable number of distinctive black bodies at the anterior half of the cell .
the cells of b. bacati had two heterodynamic flagella that were inserted subapically within a depression.
the longer anterior  flagellum extended forward and continuously probed the substrate during 'gliding' movements ; periodically, the tip of the anterior flagellum would adhere to the substrate and abruptly drag the cell forward.
the recurrent  flagellum was slightly longer than the cell body and trailed freely beneath the cell.
the cells of b. bacati were plastic and capable of rhythmic deformations ranging from contracted to relaxed states that were reminiscent of "euglenoid movement" .
the cells divided from anterior to posterior along the longitudinal axis .
cyst formation or sexual reproduction was not observed.
cells of b. bacati were found all year round, although the abundance of this species decreased significantly during the winter months.
cell surface
the cell surface of b. bacati was covered with two different morphotypes of episymbiotic bacteria:  more abundant rod shaped episymbionts and  spherical-shaped episymbionts .
the rod-shaped episymbionts were 3- <dig> μm long and were arranged in bands, about  <dig> μm wide, along the longitudinal axis of the host cell .
these bands peeled off when the host cell deteriorated.
the longitudinal bands of rod-shaped episymbionts were separated and defined by single or double rows of spherical episymbionts, each about  <dig>  μm in diameter .
these longitudinal rows usually extended nearly the entire length of the host cell and were helically organized when the host cells were in a contracted state .
the rod-shaped episymbionts were connected to the plasma membrane of the host by a glycocalyx-like material .
the spherical-shaped episymbionts were attached to the host within a corresponding concavity in the host plasma membrane .
the spherical-shaped episymbionts were highly organized and possessed an extrusive apparatus consisting of an apical "operculum" and a tightly coiled internal thread around a densely stained core .
the coiled thread was capable of rapid discharge through an apical pore when disturbed during chemical fixation for electron microscopy ; the densely stained core was discharged first, and the coiled thread followed .
the ultrastructure of the host cell surface, beneath the episymbionts, consisted of a plasma membrane that was organized into a repeated series of s-shaped folds  , a thin layer of glycoprotein, and a corset of microtubules .
the longitudinal rows of spherical-shaped episymbionts were associated with the troughs of the s-shaped folds .
the raised articulation zones between the s-shaped folds were visible in  light micrographs of contracted cells ,  scanning electron micrographs near the posterior end of the host cell , and  transmission electron micrographs .
the corset of microtubules beneath the folds formed a continuous row and was linked together by short "arms" .
tubular cisternae of endoplasmic reticulum and a layer of double-membrane bound mitochondrion-derived organelles  were positioned immediately below the superficial corset of microtubules .
the mitochondrion-derived organelles contained a granular matrix and none or very few cristae per tem profile .
there was no evidence of kinetoplast-like inclusions or any other kind of packed dna within the matrix of the mitochondrion-derived organelles.
the cytoplasm of the host cell was highly vacuolated and contained clusters of intracellular bacteria within vacuoles .
batteries of tubular extrusomes, ranging from only a few to several dozen, were also present within the host cytoplasm .
the extrusomes were circular in cross-section and had a densely stained outer region that surrounded a lighter, granular core; a cruciform element was observed in cross-section of some extrusomes .
the extrusomes were approximately  <dig> μm long, and many of them were positioned immediately beneath the raised articulation zones between the s-shaped surface folds .
nucleus, c-shaped rod apparatus, cytostomal funnel and vestibulum
the nucleus of b. bacati was positioned in the anterior half of the cell and had permanently condensed chromosomes .
the nucleus was also closely linked to a robust rod apparatus .
serial sections through the entire nucleus demonstrated that a c-shaped system of rods formed a nearly complete ring around an indented nucleus .
the c-shaped system of rods consisted of two main elements:  a main rod that was nestled against the indented nucleus  and  a folded accessory rod that was pressed tightly against the outer side of the main rod for most of its length.
we refer to this two-parted arrangement as the "c-shaped rod apparatus" .
the main rod was composed of a dense cluster of parallel lamellae that often appeared corrugated, while the accessory rod was composed of striated fibres  .
granular bodies of approximately  <dig> nm in diameter were observed in the spaces between the parallel lamellae of the main rod .
the ventral side of the main rod was embedded in an amorphous matrix that became thinner toward the posterior end of the cell, until it disappeared altogether .
a single row of longitudinal microtubules lined the external side of the main rod, which delimited the boundary between the main rod and the accessory rod for most of their length .
the anterior ends of both c-shaped rods terminated near the antero-ventral region of the nucleus .
the posterior end of the main rod was positioned within the posterior region of a feeding pocket .
this feeding pocket merged together with the flagellar pocket and formed a common subapical concavity in the cell or a "vestibulum" .
a novel "cytostomal funnel" was positioned at the junction, and therefore demarcated the boundary, between the feeding pocket and the flagellar pocket .
the cytostomal funnel was an anterior extension of the posterior end of the accessory rod that eventually opened within the subapical vestibulum .
some microtubules associated with the posterior end of the accessory rod also extended toward the ventral side of the cell and appeared to become continuous with the  microtubules that reinforced the flagellar pocket .
the posterior region of the feeding pocket also contained a "congregated globular structure"  that was associated with the posterior end of the main rod .
the posterior end of the folded accessory rod became more robust as the serial sections moved from the posterior end of the feeding pocket toward the posterior end of the cell .
the posterior end of the folded accessory rod was initially positioned between the feeding apparatus and the flagellar apparatus; the accessory rod then gradually became more robust and more tightly associated with the main rod as both of the rods migrated around the posterior side of the nucleus and toward the dorsal side of the nucleus .
moreover, as the sections continued posteriorly, the feeding pocket and the cgs that surrounded the main rod diminished, and ultimately only the main rod and the accessory rod remained .
serial sections through the anterior region of the nucleus, moving from anterior to posterior, demonstrated the c-shaped curvature of the rod apparatus .
these sections also demonstrated how the anterior ends of both the main rod and the accessory rod terminate on the ventral side of the indented nucleus near the vestibulum .
similarly, serial sections through the posterior region of the nucleus, moving from anterior to posterior, demonstrated the c-shaped curvature of the rod apparatus and its relationship to the indented nucleus .
flagellar root system
two flagella emerged from the base of the flagellar pocket .
each flagellum had a paraxial rod  in addition to the 9+ <dig> arrangement of microtubules forming the axoneme .
the pr in the dorsal flagellum  had a whorled disposition, whereas the pr of the ventral flagellum  had a lattice-like arrangement of parallel fibres .
no mastigonemes were observed on either flagellum .
the dorsal basal body contained a long opaque core .
both basal bodies were approximately  <dig>  μm long and were linked by a connecting fibre  .
a cartwheel structure was present at the proximal end of both basal bodies .
two accessory basal bodies  were observed on the ventral side of the db and the dorsal side of the vb .
the flagellar root system is described here from the proximal to the distal end of the basal bodies as viewed from the anterior end of the cell.
the basal bodies were associated with three asymmetrically arranged flagellar roots.
a dorsal root  originated from the dorsal-right side of the db  and was formed of approximately six microtubules .
a ventral root  connected to the dorsal-right side of the ventral basal body  and was comprised initially of four microtubules .
an intermediate root , originally formed of about eight microtubules , emerged from the left side of the vb .
the ventral root and the intermediate roots ultimately fused, forming a continuous vr-ir row of microtubules around the flagellar pocket .
a band of dorsal microtubules , not directly associated to the basal bodies, lined the dorsal side of the flagellar pocket .
toward the anterior end of the cell, the number of microtubules increased one by one, until the band reached the dorsal root .
the dmt and the dr eventually fused and formed a single band of microtubules around the flagellar pocket .
the dr and vr were associated with two electron dense bodies that elongated to form a dorsal lamina  and a ventral lamina , respectively .
both laminae extended anteriorly and ended up reinforcing the walls of the flagellar pocket .
the dr, together with the dl, supported the dorsal-left side of the pocket, and the dmt supported the dorsal-right side.
the vr - reinforced by the vl - lined the ventral side of the pocket and was in contact with the ir that lined the ventral-left side of the flagellar pocket.
the microtubules of the dmt and the vr became part of the elements forming the cytostomal funnel and accessory rod , and both the dr and the ir became part of the sheet of microtubules underlining the plasma membrane of the entire cell.
molecular phylogenetic position
in order to infer the phylogenetic position of b. bacati, we pcr-amplified and sequenced the nearly complete ssu rdna gene  from two independent isolates.
the sequences contained expansions typical of euglenozoan ssu rdna genes.
first, we carried out a 40-taxon maximum likelihood  analysis that included sequences representing all of the major groups of eukaryotes; the resulting phylogeny showed b. bacati grouped strongly within the euglenozoa .
a second analysis included  <dig> taxa representing all of the major lineages of euglenozoans.
the phylogenetic analyses showed that the euglenozoan sequences clustered in five main subgroups with high statistical support :  a kinetoplastid clade,  a diplonemid clade,  a bacteriovorous euglenid clade,  a eukaryovorous + phototrophic euglenid clade and  the symbiontida, a newly named clade that includes calkinsia aureus and several environmental sequences.
bihospites bacati clustered with the symbiontida with extremely high statistical support , as the sister lineage to the rest of this group.
calkinsia aureus branched next within the symbiontida and formed the sister lineage to several environmental sequences .
however, the relationship of the symbiontida to the other main subgroups within the euglenozoa was unclear.
discussion
bihospites bacati n. gen et sp.
possesses all three synapomorphies that unify the euglenozoa: a tripartite flagellar root system, heteromorphic paraxial rods and tubular extrusomes.
concordantly, our analyses of ssu rdna sequences clearly places b. bacati within the euglenozoa, specifically within the symbiontida.
several studies based on environmental sequences indicated the existence of a novel rdna clade of euglenozoans  <cit> .
however, the symbiontida was proposed after the ultrastructural description and molecular phylogeny of c. aureus strongly grouped this species with these environmental sequences, as a distinct subgroup within the euglenozoa  <cit> .
nonetheless, it was not clear in that study whether the symbiontida was a new clade of euglenozoans or a subclade within one of the three previously recognized members of the euglenozoa .
our comprehensive characterization of b. bacati sheds considerable light onto this question.
remnants of pellicle strips
bihospites bacati possesses a cell surface consisting of s-shaped folds, microtubules and endoplasmic reticulum that is similar to the pellicle of s-shaped strips found in euglenids.
in most photosynthetic euglenids, the pellicle strips usually consist of a robust proteinaceous frame that supports and maintains the shape of the cell, even during euglenoid movement  <cit> .
however, like in most phagotrophic euglenids, there is no robust proteinaceous frame in b. bacati.
articulation zones between strips in the euglenid pellicle function as 'slipping points' around which the pellicle can change shape rather freely; moreover, the relative number of strips in each euglenid species reflects phylogenetic relationships and the degree of cell plasticity  <cit> .
due to the extreme flexibility of the cell surface in b. bacati, it was not possible to determine an exact number of s-shaped folds in the cell surface.
nonetheless, the microtubular corset in most euglenids is regularly interrupted, thus forming groups of a few microtubules associated with each pellicle strip, the number of which varies between species  <cit> .
by contrast, the microtubules beneath the plasma membrane in b. bacati form a continuous corset over the entire cell, much like that found in several phagotrophic euglenids  and in symbiontids .
a novel feeding apparatus consisting of rods
bihospites bacati possesses a well-developed c-shaped rod apparatus consisting of a main rod and an associated accessory rod.
several heterotrophic euglenids  <cit> , and some species of diplonemids  <cit> , have been described with feeding apparatuses consisting of two main rods; some species also have corresponding accessory rods  or have a branched rod that gives the appearance of three main rods .
nonetheless, there are several differences between these rods and those described here for b. bacati.
firstly, b. bacati only has one main rod and one folded accessory rod; this configuration has never been described so far.
secondly, the vast majority of this apparatus tightly encircles the nucleus in a c-shaped fashion, the functional significance of which is totally unclear.
the straight rods in euglenids support and line a conspicuous feeding pocket, whereas the feeding pocket in b. bacati only associates with the posterior end of the c-shaped rod apparatus.
thirdly, the main c-shaped rod in b. bacati is formed by a highly novel arrangement of tightly packed lamellae, and only a single row of microtubules originating from the vr separates the main c-shaped rod from the folded accessory rod.
this row of microtubules demarcates the end of each lamella in the main rod.
in all of the previously described euglenozoan species, different rods are formed by different proportions of amorphous material  and microtubules originating from the ventral root of the ventral basal body.
fourthly, the posterior terminus of the accessory rod in b. bacati participates in the formation of a novel cytostomal funnel that extends anteriorly and merges with the subapical vestibulum.
the cytostomal funnel presumably closes the connection between the flagellar pocket and the vestibulum during feeding.
although the cytostomal funnel in b. bacati is likely homologous to the "vanes" described in several different phagotrophic euglenids, the unusual ultrastructural features of b. bacati made this inference somewhat tenuous.
nonetheless, the additional "congregated globular structure"  at the posterior end of the main rod in b. bacati is also present in calkinsia aureus  <cit> .
however, the feeding apparatus in c. aureus lacks conspicuous rods  and consists mainly of a feeding pocket reinforced by microtubules from the vr, similar to the mtr pockets of other euglenozoans .
overall, the c-shaped rod apparatus in b. bacati appears to contain some homologous subcomponents with phagotrophic euglenozoans , but, as highlighted above, this apparatus is novel in most respects.
the presence of a highly plastic cell surface, an elaborate feeding apparatus, and brownish bodies, reminiscent of food vacuoles, suggests that b. bacati is capable of engulfing large prey cells such as other eukaryotes  <cit> ; however, this species was never directly observed preying on  microeukaryotic cells present in the environment.
nonetheless, the presence of intracellular bacteria surrounded by vacuoles near the feeding pocket indicates that b. bacati actively feeds on bacteria.
it is also possible that b. bacati feeds on the rod shaped episymbiotic bacteria that grow over the host surface and into the subapical vestibulum.
extrusomes
tubular extrusomes are present in several members of the euglenozoa  <cit>  and constitute a synapomorphy for the group.
among the symbiontida, c. aureus has tubular extrusomes clustered in a single large battery that is longitudinally arranged and anchored to a novel "extrusomal pocket"  <cit> .
although bihospites bacati also possesses tubular extrusomes, these organelles are not organized as a single battery.
the extrusomes in b. bacati are arranged in several smaller clusters that are distributed in different places throughout the superficial cytoplasm; solitary extrusomes are organized consecutively beneath the articulation zones of the s-shaped pellicular folds or "strips".
a similar arrangement of tubular extrusomes has also been observed in p. mariagerensis  <cit> .
episymbiotic bacteria
several distantly related species of euglenozoans have been described with episymbiotic bacteria.
these euglenozoans are usually phagotrophs that live in oxygen-depletd to anoxic marine environments, such as that in which b. bacati thrives  <cit> .
however, two species of euglenids living in well-oxygenated, freshwater environments have also been described as having episymbiotic bacteria: the phototroph euglena helicoideus  <cit> , and the phagotroph dylakosoma pelophilum  <cit> .
the episymbionts so far encountered in euglenozoans are either rod-shaped  or spherical-shaped .
bihospites bacati, however, is the first euglenozoan described with both morphotypes of episymbionts.
hypotheses about the role of rod-shaped bacteria in symbiotic relationships with eukaryotic hosts usually emphasize commensalism, where the bacteria benefit from metabolic byproducts secreted by the host  <cit> .
it has also been proposed that the rod-shaped bacteria are chemoautotrophic sulphur or methanogenic-oxydizers and form a mutualistic relationship with the host  <cit> , whereby the host provides anchorage for the bacteria and the bacteria detoxify the immediate environment for the host  <cit> .
the episymbiotic bacteria may also serve as a food-source for the host, as has been observed in one ciliate  <cit> .
spherical episymbiotic bacteria have been reported in one other euglenozoan based only on light microscopy: the freshwater euglenid d. pelophilum  <cit> .
however, this species has so far been poorly described and morphological characteristics of the bacteria are very difficult to evaluate; it was reported that the bacteria on the surface of d. pelophilum are  <dig> μm in diameter, twice the size of those in b. bacati.
spherical episymbiotic bacteria that are nearly identical at the ultrastructural level to those we describe here on b. bacati have been demonstrated on one species of hypotrich ciliate isolated from tidal pools  <cit> .
molecular phylogenetic evidence demonstrates that these episymbionts, called "epixenosomes", are novel lineages of verrucomicrobial bacteria, and experiments indicate that the extrusive nature of the spherical episymbionts function in defense against predators  <cit> .
therefore, these episymbionts improve the comparative context for understanding the origin of different types of extrusive organelles in different lineages of eukaryotes .
a more comprehensive examination and discussion of the biology and origins of the epixenosomes in b. bacati have been incorporated into a companion paper currently in preparation for publication .
the identity and composition of the symbiontida
molecular phylogenetic analyses using ssu sequences place b. bacati as the earliest diverging branch within the symbiontida.
the symbiontida are anaerobic and microaerophilic euglenozoans covered with rod-shaped bacteria that are in close association with a superficial layer of mitochondrion-derived organelles with reduced or absent cristae; accordingly, it was predicted that rod-shaped episymbionts are present in most  members of the group  <cit> .
the morphology of b. bacati is concordant with this description, reinforcing the interpretation that the presence of episymbiotic bacteria is a shared derived character of the most recent ancestor of the symbiontida.
this hypothesis is more robustly corroborated when we consider that b. bacati and c. aureus form a paraphyletic assemblage near the origin of the symbiontida.
in other words, episymbiotic bacteria are no longer a character known only in a single lineage within this group.
given this context, current ultrastructural data indicate that p. mariagerensis is also a member of the symbiontida   <cit> .
this inference, however, needs to be examined more carefully with an ultrastructural characterization of the flagellar apparatus and feeding apparatus in p. mariagerensis and with molecular phylogenetic data from the host and the episymbionts.
the presence of episymbiotic bacteria and the superficial distribution of mitochondria with reduced cristae in b. bacati, c. aureus and p. mariagerensis indicate a mutualistic relationship that enabled both lineages to diversify within low-oxygen environments.
determining whether the episymbionts on b. bacati, c. aureus and other symbiontids are closely related will more robustly establish the identity and composition of the clade and potentially reveal co-evolutionary patterns between the symbionts and the hosts.
the geographic distribution of c. aureus and b. bacati  suggests that the symbiontida is more widespread and diverse than currently known.
this view is supported by the existence of related environmental sequences originating from venezuela, denmark and norway  <cit> .
moreover, an organism with striking morphological resemblance to b. bacati has been previously observed in the wadden sea, germany,  <cit> .
more comprehensive sampling of anoxic and low-oxygen sediments around the world will shed considerable light on the abundances and ecological significance of this enigmatic group of euglenozoans.
CONCLUSIONS
we described and characterized a novel flagellate from micro-aerobic marine sand: bihospites bacati n. gen. et sp.
both comparative ultrastructure and molecular phylogenetic analyses strongly support the placement of b. bacati with the euglenozoa and, more specifically, as a new member of the symbiontida.
an early diverging position of b. bacati within the symbiontida is consistent with the presence of morphological features that are transitional between those found in c. aureus and phagotrophic euglenids:  a cell surface with strip-like s-shaped folds but lacking the proteinaceous frames of the euglenid pellicle,  a compact but robust rod-based feeding apparatus, and  a dense community of rod-shaped episymbiotic bacteria on the cell surface but without the elaborate extracellular matrix of c. aureus.
therefore, the molecular phylogenetic position and suite of intermediate ultrastructural features in b. bacati suggest that the most recent ancestor of the symbiontida descended from phagotrophic euglenids.
although the close association of rod-shaped episymbiotic bacteria with the underlying mitochondria is a shared feature of symbiontids, the presence of extrusive verrucomicrobial episymbionts in b. bacati is highly unusual.
these rapid-firing episymbionts could provide critical context for understanding the origin of several different types of extrusive organelles in eukaryotes, and their discovery on this novel euglenozoan lineage underscores how little we know about the diverse symbiotic communities present in marine benthic environments.

 the euglenozoa is a diverse group of single-celled eukaryotes consisting of three main subgroups: euglenids, kinetoplastids and diplonemids. euglenids are united by the presence of a distinctive pellicle, a superficial system formed by four major components: the plasma membrane, a pattern of repeating proteinaceous strips that run along the length of the cell, subtending microtubules and tubular cisternae of endoplasmic reticulum. the group is widely known for its photosynthetic members, but the majority of the species are heterotrophic. photosynthetic euglenids evolved from phagotrophic ancestors with a complex feeding apparatus and a large number of pellicle strips that facilitate a characteristic peristaltic cell movement called "euglenoid movement". this combination of characters allows phagotrophic euglenids to engulf large prey cells, such as eukaryotic algae, which eventually led to the acquisition of chloroplasts via secondary endosymbiosis. euglenids are closely related to kinetoplastids and diplonemids. kinetoplastids are united by the presence of a mitochondrial inclusion of distinctively arranged dna molecules, called a kinetoplast or kdna. kinetoplastids and euglenids share several morphological features, such as flagella with hairs and heteromorphic paraxial rods and mitochondria with paddle-shaped cristae. diplonemids, on the other hand, possess a large mitochondrion with flattened cristae and apparently lack flagellar hairs. the monophyly of the euglenozoa has been established on the basis of both molecular phylogenetic analyses and the following morphological synapomorphies: a tripartite flagellar root system, presence of heteromorphic paraxial rods and tubular extrusomes. environmental sequencing of oxygen depleted sediments around the world has shown that these habitats harbour a vast and unknown diversity of microbial lineages. phylogenetic analyses of these data have helped demonstrate the existence of several novel lineages associated with many different eukaryotic supergroups. although these types of analyses are very effective in revealing the actual diversity of microbes living in a particular environment, these approaches also generate vast amounts of "orphan" data that cannot be linked directly to organisms known from comparative morphology. nonetheless, some of the environmental sequences recovered from oxygen depleted environments cluster with euglenozoans in phylogenetic analyses but with no clear position within the group. other studies have explored and characterized the microbial diversity in oxygen-depleted environments using microscopical approaches. this research has shown that a reoccurring feature of euglenozoans living in low oxygen environments is the presence of episymbiotic bacteria on the cell surface. here, we report on a highly unusual euglenozoan isolated from oxygen depleted marine sediments that is covered with two very different morphotypes of episymbionts. we characterized this lineage with light microscopy, sem, comprehensive tem, and molecular phylogenetic analyses of ssu rdna sequences. our data demonstrate that this organism is the earliest diverging member of the symbiontida, which is an emerging subclade of euglenozoans composed of anaerobic and microaerophilic flagellates with a superficial layer of mitochondrion-derived organelles that associates closely with a uniform layer of episymbiotic bacteria. moreover, the comparative ultrastructural data from this novel lineage sheds considerable light onto the phylogenetic position of the symbiontida, as a whole, within the euglenozoa.  general morphology the cells of bihospites bacati n. gen. et sp. were elongated with a somewhat rounded posterior end and were 40- μm long and 15- μm wide. the cells contained a brownish body near the posterior end of the cell and a variable number of distinctive black bodies at the anterior half of the cell. the cells of b. bacati had two heterodynamic flagella that were inserted subapically within a depression. the longer anterior flagellum extended forward and continuously probed the substrate during'gliding' movements; periodically, the tip of the anterior flagellum would adhere to the substrate and abruptly drag the cell forward. the recurrent flagellum was slightly longer than the cell body and trailed freely beneath the cell. the cells of b. bacati were plastic and capable of rhythmic deformations ranging from contracted to relaxed states that were reminiscent of "euglenoid movement". the cells divided from anterior to posterior along the longitudinal axis. cyst formation or sexual reproduction was not observed. cells of b. bacati were found all year round, although the abundance of this species decreased significantly during the winter months. cell surface the cell surface of b. bacati was covered with two different morphotypes of episymbiotic bacteria: more abundant rod shaped episymbionts and spherical-shaped episymbionts. the rod-shaped episymbionts were 3- μm long and were arranged in bands, about μm wide, along the longitudinal axis of the host cell. these bands peeled off when the host cell deteriorated. the longitudinal bands of rod-shaped episymbionts were separated and defined by single or double rows of spherical episymbionts, each about μm in diameter. these longitudinal rows usually extended nearly the entire length of the host cell and were helically organized when the host cells were in a contracted state. the rod-shaped episymbionts were connected to the plasma membrane of the host by a glycocalyx-like material. the spherical-shaped episymbionts were attached to the host within a corresponding concavity in the host plasma membrane. the spherical-shaped episymbionts were highly organized and possessed an extrusive apparatus consisting of an apical "operculum" and a tightly coiled internal thread around a densely stained core. the coiled thread was capable of rapid discharge through an apical pore when disturbed during chemical fixation for electron microscopy; the densely stained core was discharged first, and the coiled thread followed. the ultrastructure of the host cell surface, beneath the episymbionts, consisted of a plasma membrane that was organized into a repeated series of s-shaped folds, a thin layer of glycoprotein, and a corset of microtubules. the longitudinal rows of spherical-shaped episymbionts were associated with the troughs of the s-shaped folds. the raised articulation zones between the s-shaped folds were visible in light micrographs of contracted cells, scanning electron micrographs near the posterior end of the host cell, and transmission electron micrographs. the corset of microtubules beneath the folds formed a continuous row and was linked together by short "arms". tubular cisternae of endoplasmic reticulum and a layer of double-membrane bound mitochondrion-derived organelles were positioned immediately below the superficial corset of microtubules. the mitochondrion-derived organelles contained a granular matrix and none or very few cristae per tem profile. there was no evidence of kinetoplast-like inclusions or any other kind of packed dna within the matrix of the mitochondrion-derived organelles. the cytoplasm of the host cell was highly vacuolated and contained clusters of intracellular bacteria within vacuoles. batteries of tubular extrusomes, ranging from only a few to several dozen, were also present within the host cytoplasm. the extrusomes were circular in cross-section and had a densely stained outer region that surrounded a lighter, granular core; a cruciform element was observed in cross-section of some extrusomes. the extrusomes were approximately μm long, and many of them were positioned immediately beneath the raised articulation zones between the s-shaped surface folds. nucleus, c-shaped rod apparatus, cytostomal funnel and vestibulum the nucleus of b. bacati was positioned in the anterior half of the cell and had permanently condensed chromosomes. the nucleus was also closely linked to a robust rod apparatus. serial sections through the entire nucleus demonstrated that a c-shaped system of rods formed a nearly complete ring around an indented nucleus. the c-shaped system of rods consisted of two main elements: a main rod that was nestled against the indented nucleus and a folded accessory rod that was pressed tightly against the outer side of the main rod for most of its length. we refer to this two-parted arrangement as the "c-shaped rod apparatus". the main rod was composed of a dense cluster of parallel lamellae that often appeared corrugated, while the accessory rod was composed of striated fibres. granular bodies of approximately nm in diameter were observed in the spaces between the parallel lamellae of the main rod. the ventral side of the main rod was embedded in an amorphous matrix that became thinner toward the posterior end of the cell, until it disappeared altogether. a single row of longitudinal microtubules lined the external side of the main rod, which delimited the boundary between the main rod and the accessory rod for most of their length. the anterior ends of both c-shaped rods terminated near the antero-ventral region of the nucleus. the posterior end of the main rod was positioned within the posterior region of a feeding pocket. this feeding pocket merged together with the flagellar pocket and formed a common subapical concavity in the cell or a "vestibulum". a novel "cytostomal funnel" was positioned at the junction, and therefore demarcated the boundary, between the feeding pocket and the flagellar pocket. the cytostomal funnel was an anterior extension of the posterior end of the accessory rod that eventually opened within the subapical vestibulum. some microtubules associated with the posterior end of the accessory rod also extended toward the ventral side of the cell and appeared to become continuous with the microtubules that reinforced the flagellar pocket. the posterior region of the feeding pocket also contained a "congregated globular structure" that was associated with the posterior end of the main rod. the posterior end of the folded accessory rod became more robust as the serial sections moved from the posterior end of the feeding pocket toward the posterior end of the cell. the posterior end of the folded accessory rod was initially positioned between the feeding apparatus and the flagellar apparatus; the accessory rod then gradually became more robust and more tightly associated with the main rod as both of the rods migrated around the posterior side of the nucleus and toward the dorsal side of the nucleus. moreover, as the sections continued posteriorly, the feeding pocket and the cgs that surrounded the main rod diminished, and ultimately only the main rod and the accessory rod remained. serial sections through the anterior region of the nucleus, moving from anterior to posterior, demonstrated the c-shaped curvature of the rod apparatus. these sections also demonstrated how the anterior ends of both the main rod and the accessory rod terminate on the ventral side of the indented nucleus near the vestibulum. similarly, serial sections through the posterior region of the nucleus, moving from anterior to posterior, demonstrated the c-shaped curvature of the rod apparatus and its relationship to the indented nucleus. flagellar root system two flagella emerged from the base of the flagellar pocket. each flagellum had a paraxial rod in addition to the 9+ arrangement of microtubules forming the axoneme. the pr in the dorsal flagellum had a whorled disposition, whereas the pr of the ventral flagellum had a lattice-like arrangement of parallel fibres. no mastigonemes were observed on either flagellum. the dorsal basal body contained a long opaque core. both basal bodies were approximately μm long and were linked by a connecting fibre. a cartwheel structure was present at the proximal end of both basal bodies. two accessory basal bodies were observed on the ventral side of the db and the dorsal side of the vb. the flagellar root system is described here from the proximal to the distal end of the basal bodies as viewed from the anterior end of the cell. the basal bodies were associated with three asymmetrically arranged flagellar roots. a dorsal root originated from the dorsal-right side of the db and was formed of approximately six microtubules. a ventral root connected to the dorsal-right side of the ventral basal body and was comprised initially of four microtubules. an intermediate root, originally formed of about eight microtubules, emerged from the left side of the vb. the ventral root and the intermediate roots ultimately fused, forming a continuous vr-ir row of microtubules around the flagellar pocket. a band of dorsal microtubules, not directly associated to the basal bodies, lined the dorsal side of the flagellar pocket. toward the anterior end of the cell, the number of microtubules increased one by one, until the band reached the dorsal root. the dmt and the dr eventually fused and formed a single band of microtubules around the flagellar pocket. the dr and vr were associated with two electron dense bodies that elongated to form a dorsal lamina and a ventral lamina, respectively. both laminae extended anteriorly and ended up reinforcing the walls of the flagellar pocket. the dr, together with the dl, supported the dorsal-left side of the pocket, and the dmt supported the dorsal-right side. the vr - reinforced by the vl - lined the ventral side of the pocket and was in contact with the ir that lined the ventral-left side of the flagellar pocket. the microtubules of the dmt and the vr became part of the elements forming the cytostomal funnel and accessory rod, and both the dr and the ir became part of the sheet of microtubules underlining the plasma membrane of the entire cell. molecular phylogenetic position in order to infer the phylogenetic position of b. bacati, we pcr-amplified and sequenced the nearly complete ssu rdna gene from two independent isolates. the sequences contained expansions typical of euglenozoan ssu rdna genes. first, we carried out a 40-taxon maximum likelihood analysis that included sequences representing all of the major groups of eukaryotes; the resulting phylogeny showed b. bacati grouped strongly within the euglenozoa. a second analysis included taxa representing all of the major lineages of euglenozoans. the phylogenetic analyses showed that the euglenozoan sequences clustered in five main subgroups with high statistical support: a kinetoplastid clade, a diplonemid clade, a bacteriovorous euglenid clade, a eukaryovorous + phototrophic euglenid clade and the symbiontida, a newly named clade that includes calkinsia aureus and several environmental sequences. bihospites bacati clustered with the symbiontida with extremely high statistical support, as the sister lineage to the rest of this group. calkinsia aureus branched next within the symbiontida and formed the sister lineage to several environmental sequences. however, the relationship of the symbiontida to the other main subgroups within the euglenozoa was unclear. discussion bihospites bacati n. gen et sp. possesses all three synapomorphies that unify the euglenozoa: a tripartite flagellar root system, heteromorphic paraxial rods and tubular extrusomes. concordantly, our analyses of ssu rdna sequences clearly places b. bacati within the euglenozoa, specifically within the symbiontida. several studies based on environmental sequences indicated the existence of a novel rdna clade of euglenozoans. however, the symbiontida was proposed after the ultrastructural description and molecular phylogeny of c. aureus strongly grouped this species with these environmental sequences, as a distinct subgroup within the euglenozoa. nonetheless, it was not clear in that study whether the symbiontida was a new clade of euglenozoans or a subclade within one of the three previously recognized members of the euglenozoa. our comprehensive characterization of b. bacati sheds considerable light onto this question. remnants of pellicle strips bihospites bacati possesses a cell surface consisting of s-shaped folds, microtubules and endoplasmic reticulum that is similar to the pellicle of s-shaped strips found in euglenids. in most photosynthetic euglenids, the pellicle strips usually consist of a robust proteinaceous frame that supports and maintains the shape of the cell, even during euglenoid movement. however, like in most phagotrophic euglenids, there is no robust proteinaceous frame in b. bacati. articulation zones between strips in the euglenid pellicle function as'slipping points' around which the pellicle can change shape rather freely; moreover, the relative number of strips in each euglenid species reflects phylogenetic relationships and the degree of cell plasticity. due to the extreme flexibility of the cell surface in b. bacati, it was not possible to determine an exact number of s-shaped folds in the cell surface. nonetheless, the microtubular corset in most euglenids is regularly interrupted, thus forming groups of a few microtubules associated with each pellicle strip, the number of which varies between species. by contrast, the microtubules beneath the plasma membrane in b. bacati form a continuous corset over the entire cell, much like that found in several phagotrophic euglenids and in symbiontids. a novel feeding apparatus consisting of rods bihospites bacati possesses a well-developed c-shaped rod apparatus consisting of a main rod and an associated accessory rod. several heterotrophic euglenids, and some species of diplonemids, have been described with feeding apparatuses consisting of two main rods; some species also have corresponding accessory rods or have a branched rod that gives the appearance of three main rods. nonetheless, there are several differences between these rods and those described here for b. bacati. firstly, b. bacati only has one main rod and one folded accessory rod; this configuration has never been described so far. secondly, the vast majority of this apparatus tightly encircles the nucleus in a c-shaped fashion, the functional significance of which is totally unclear. the straight rods in euglenids support and line a conspicuous feeding pocket, whereas the feeding pocket in b. bacati only associates with the posterior end of the c-shaped rod apparatus. thirdly, the main c-shaped rod in b. bacati is formed by a highly novel arrangement of tightly packed lamellae, and only a single row of microtubules originating from the vr separates the main c-shaped rod from the folded accessory rod. this row of microtubules demarcates the end of each lamella in the main rod. in all of the previously described euglenozoan species, different rods are formed by different proportions of amorphous material and microtubules originating from the ventral root of the ventral basal body. fourthly, the posterior terminus of the accessory rod in b. bacati participates in the formation of a novel cytostomal funnel that extends anteriorly and merges with the subapical vestibulum. the cytostomal funnel presumably closes the connection between the flagellar pocket and the vestibulum during feeding. although the cytostomal funnel in b. bacati is likely homologous to the "vanes" described in several different phagotrophic euglenids, the unusual ultrastructural features of b. bacati made this inference somewhat tenuous. nonetheless, the additional "congregated globular structure" at the posterior end of the main rod in b. bacati is also present in calkinsia aureus. however, the feeding apparatus in c. aureus lacks conspicuous rods and consists mainly of a feeding pocket reinforced by microtubules from the vr, similar to the mtr pockets of other euglenozoans. overall, the c-shaped rod apparatus in b. bacati appears to contain some homologous subcomponents with phagotrophic euglenozoans, but, as highlighted above, this apparatus is novel in most respects. the presence of a highly plastic cell surface, an elaborate feeding apparatus, and brownish bodies, reminiscent of food vacuoles, suggests that b. bacati is capable of engulfing large prey cells such as other eukaryotes; however, this species was never directly observed preying on microeukaryotic cells present in the environment. nonetheless, the presence of intracellular bacteria surrounded by vacuoles near the feeding pocket indicates that b. bacati actively feeds on bacteria. it is also possible that b. bacati feeds on the rod shaped episymbiotic bacteria that grow over the host surface and into the subapical vestibulum. extrusomes tubular extrusomes are present in several members of the euglenozoa and constitute a synapomorphy for the group. among the symbiontida, c. aureus has tubular extrusomes clustered in a single large battery that is longitudinally arranged and anchored to a novel "extrusomal pocket". although bihospites bacati also possesses tubular extrusomes, these organelles are not organized as a single battery. the extrusomes in b. bacati are arranged in several smaller clusters that are distributed in different places throughout the superficial cytoplasm; solitary extrusomes are organized consecutively beneath the articulation zones of the s-shaped pellicular folds or "strips". a similar arrangement of tubular extrusomes has also been observed in p. mariagerensis. episymbiotic bacteria several distantly related species of euglenozoans have been described with episymbiotic bacteria. these euglenozoans are usually phagotrophs that live in oxygen-depletd to anoxic marine environments, such as that in which b. bacati thrives. however, two species of euglenids living in well-oxygenated, freshwater environments have also been described as having episymbiotic bacteria: the phototroph euglena helicoideus, and the phagotroph dylakosoma pelophilum. the episymbionts so far encountered in euglenozoans are either rod-shaped or spherical-shaped. bihospites bacati, however, is the first euglenozoan described with both morphotypes of episymbionts. hypotheses about the role of rod-shaped bacteria in symbiotic relationships with eukaryotic hosts usually emphasize commensalism, where the bacteria benefit from metabolic byproducts secreted by the host. it has also been proposed that the rod-shaped bacteria are chemoautotrophic sulphur or methanogenic-oxydizers and form a mutualistic relationship with the host, whereby the host provides anchorage for the bacteria and the bacteria detoxify the immediate environment for the host. the episymbiotic bacteria may also serve as a food-source for the host, as has been observed in one ciliate. spherical episymbiotic bacteria have been reported in one other euglenozoan based only on light microscopy: the freshwater euglenid d. pelophilum. however, this species has so far been poorly described and morphological characteristics of the bacteria are very difficult to evaluate; it was reported that the bacteria on the surface of d. pelophilum are μm in diameter, twice the size of those in b. bacati. spherical episymbiotic bacteria that are nearly identical at the ultrastructural level to those we describe here on b. bacati have been demonstrated on one species of hypotrich ciliate isolated from tidal pools. molecular phylogenetic evidence demonstrates that these episymbionts, called "epixenosomes", are novel lineages of verrucomicrobial bacteria, and experiments indicate that the extrusive nature of the spherical episymbionts function in defense against predators. therefore, these episymbionts improve the comparative context for understanding the origin of different types of extrusive organelles in different lineages of eukaryotes. a more comprehensive examination and discussion of the biology and origins of the epixenosomes in b. bacati have been incorporated into a companion paper currently in preparation for publication. the identity and composition of the symbiontida molecular phylogenetic analyses using ssu sequences place b. bacati as the earliest diverging branch within the symbiontida. the symbiontida are anaerobic and microaerophilic euglenozoans covered with rod-shaped bacteria that are in close association with a superficial layer of mitochondrion-derived organelles with reduced or absent cristae; accordingly, it was predicted that rod-shaped episymbionts are present in most members of the group. the morphology of b. bacati is concordant with this description, reinforcing the interpretation that the presence of episymbiotic bacteria is a shared derived character of the most recent ancestor of the symbiontida. this hypothesis is more robustly corroborated when we consider that b. bacati and c. aureus form a paraphyletic assemblage near the origin of the symbiontida. in other words, episymbiotic bacteria are no longer a character known only in a single lineage within this group. given this context, current ultrastructural data indicate that p. mariagerensis is also a member of the symbiontida. this inference, however, needs to be examined more carefully with an ultrastructural characterization of the flagellar apparatus and feeding apparatus in p. mariagerensis and with molecular phylogenetic data from the host and the episymbionts. the presence of episymbiotic bacteria and the superficial distribution of mitochondria with reduced cristae in b. bacati, c. aureus and p. mariagerensis indicate a mutualistic relationship that enabled both lineages to diversify within low-oxygen environments. determining whether the episymbionts on b. bacati, c. aureus and other symbiontids are closely related will more robustly establish the identity and composition of the clade and potentially reveal co-evolutionary patterns between the symbionts and the hosts. the geographic distribution of c. aureus and b. bacati suggests that the symbiontida is more widespread and diverse than currently known. this view is supported by the existence of related environmental sequences originating from venezuela, denmark and norway. moreover, an organism with striking morphological resemblance to b. bacati has been previously observed in the wadden sea, germany,. more comprehensive sampling of anoxic and low-oxygen sediments around the world will shed considerable light on the abundances and ecological significance of this enigmatic group of euglenozoans.  we described and characterized a novel flagellate from micro-aerobic marine sand: bihospites bacati n. gen. et sp. both comparative ultrastructure and molecular phylogenetic analyses strongly support the placement of b. bacati with the euglenozoa and, more specifically, as a new member of the symbiontida. an early diverging position of b. bacati within the symbiontida is consistent with the presence of morphological features that are transitional between those found in c. aureus and phagotrophic euglenids: a cell surface with strip-like s-shaped folds but lacking the proteinaceous frames of the euglenid pellicle, a compact but robust rod-based feeding apparatus, and a dense community of rod-shaped episymbiotic bacteria on the cell surface but without the elaborate extracellular matrix of c. aureus. therefore, the molecular phylogenetic position and suite of intermediate ultrastructural features in b. bacati suggest that the most recent ancestor of the symbiontida descended from phagotrophic euglenids. although the close association of rod-shaped episymbiotic bacteria with the underlying mitochondria is a shared feature of symbiontids, the presence of extrusive verrucomicrobial episymbionts in b. bacati is highly unusual. these rapid-firing episymbionts could provide critical context for understanding the origin of several different types of extrusive organelles in eukaryotes, and their discovery on this novel euglenozoan lineage underscores how little we know about the diverse symbiotic communities present in marine benthic environments. 
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. we characterized the ultrastructure and molecular phylogenetic position of b. bacati n. gen. et sp. these parallel symbioses increase the comparative context for understanding the origin of extrusive organelles in eukaryotes and underscores how little we know about the symbiotic communities of marine benthic environments. 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. 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. ultrastructural data showed that b. bacati possesses all of the euglenozoan synapomorphies. 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. we discovered and characterized a novel 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. molecular phylogenetic analyses demonstrated that this species belongs to the euglenozoa and currently branches as the earliest diverging member of the symbiontida. poorly understood but highly diverse microbial communities exist within anoxic and oxygen-depleted marine sediments. bihospites bacati n. gen. et sp.
 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- cm depth; these layers contain a rich variety of microorganisms, many of which are undescribed. we discovered and characterized a novel 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.  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: rod-shaped bacteria and 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.  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 of extrusive organelles in eukaryotes and underscores how little we know about the symbiotic communities of marine benthic environments. 