BACKGROUND
the euglenozoa is a clade of eukaryotic microorganisms with very diverse lifestyles and that tentatively falls within one of six emerging supergroups of eukaryotes, namely the "excavata"  <cit> .
most euglenozoans cluster within three major subgroups that have been established with both molecular phylogenetic analyses and combination of ultrastructural characteristics : the kinetoplastida, the euglenida and the diplonemida  <cit> .
kinetoplastids possess mitochondria with a uniquely structured genome, called "kinetoplast" dna, and the group includes both free-living phagotrophic lineages  and parasitic lineages .
euglenids possess a cytoskeleton, or "pellicle", consisting of overlapping proteinaceous strips that are arranged either longitudinally or helically, and the group includes bacteriovorous lineages , eukaryovorous lineages , osmotrophic lineages  and photosynthetic lineages .
the mitochondria of kinetoplastids and euglenids possess cristae that are distinctively discoidal in shape.
by contrast, diplonemids consist of only two genera, diplonema and rhynchopus, with sack-shaped cells, short flagella and flattened mitochondrial cristae and without kinetoplast dna, pellicle strips, and paraxonemal rods.
ultrastructural studies have also demonstrated lineages of euglenozoans that do not fall neatly within any of the three established subgroups, such as postgaardi mariagerensis, which inhabits low oxygen environments and is covered with epibiotic bacteria  <cit> .
currently, p. mariagerensis is grouped together with another poorly understood anoxic flagellate, namely calkinsia aureus, as incertae sedis within the euglenozoa  <cit> ; although molecular data is unavailable for both species, one author has chosen to classify them within a taxon called the "postgaardea"  <cit> .
c. aureus was originally collected from anoxic sediments near woods hole, ma  and described with only line drawings as a member of the euglenid family petalomonidae; this conclusion was based on the appearance of a rigid cell containing strip-like surface striations  <cit> .
however, c. aureus was subsequently collected from low-oxygen sediments in the santa barbara basin, ca  and partially studied with light and scanning electron microscopy   <cit> .
these studies demonstrated that like p. mariagerensis, c. aureus was covered with the rod-shape epibiotic bacteria, rather than pellicle strips per se.
the ultrastructure and molecular phylogenetic position of c. aureus is currently unknown.
these data are expected to help establish robust inferences about the overall diversity of euglenozoans and the ultrastructure of prokaryote-eukaryote symbioses within the group and beyond.
the main goals of this study were to characterize the ultrastructure and molecular phylogenetic position of c. aureus using small subunit  rdna sequences and transmission electron microscopy  of serially sectioned cells.
our results demonstrated that c. aureus is the first member of a novel group of anoxic euglenozoans – referred to here as the "symbiontida" – to be characterized at both the molecular and ultrastructural levels.
a companion study centered on the molecular identity and detailed ultrastructure of the epibiotic bacteria on c. aureus is currently underway.
methods
collection of organisms
calkinsia aureus was collected using a soutar box corer or mc- <dig> multi corer from the sea floor sediment  of the santa barbara basin, california, usa in september of  <dig> and june of  <dig>  sediment core samples were collected on the r/v robert gordon sproul.
some sediment samples were immediately fixed for transmission electron microscopy  with an equal volume of 4%  glutaraldehyde in  <dig>  m sodium cacodylate buffer   and stored at 4°c.
the remaining sediment samples were stored in  <dig> ml plastic tubes at 4°c and subsequently processed for light microscopy, scanning electron microscopy  and dna extraction.
light and electron microscopy
light micrographs of over  <dig> living cells were taken using a zeiss axioplan  <dig> imaging microscope and a leica dc <dig> digital chilled ccd camera.
cells of c. aureus were prepared for sem by mixing an equal volume of fixative solution containing 4%  glutaraldehyde in  <dig>  m scb  at room temperature.
the fixed cells were mounted on polycarbonate millipore filters  or glass plates coated with poly-l-lysine at room temperature for  <dig> hr.
the cells were rinsed with  <dig>  m scb and fixed in 1% osmium tetroxide for  <dig> min.
the osmium-fixed cells were then rinsed with  <dig>  m scb and dehydrated with a graded ethanol series from 30% to absolute ethanol before being critical point dried with co <dig> using a tousimis critical point dryer.
the dried cells were then coated with gold using a cressington 208hr high resolution sputter coater, and observed with a hitachi s- <dig> field emission scanning electron microscope.
cells of c. aureus prepared for tem were kept in fixative solution for two months before being individually isolated from the surrounding sediment in the sample.
isolated cells were rinsed with  <dig>  m scb  three times and then fixed in 1%  osmium tetroxide in  <dig>  m scb  at room temperature for  <dig> hr before being dehydrated through a graded series of ethanol and 100% acetone.
the dehydrated cells were then infiltrated with acetone-epon  <dig> resin mixtures and 100% resin.
individual cells were flat embedded and serial sectioned in different orientations .
ultra-thin serial sections were collected on copper, formvar-coated slot grids and stained with 2%  uranyl acetate and lead citrate  <cit>  before being observed using a hitachi h <dig> electron microscope.
a total of  <dig> micrographs from  <dig> different cells were observed.
two different media were used in an attempt to culture c. aureus: 5% of tygm- <dig>  and 5% of modified pynfh medium , diluted in anoxic and axenic seawater at 4°c.
however, the cells did not grow in either medium.
dna extraction, pcr amplification, alignment and phylogenetic analysis
twenty individual cells of c. aureus that corresponded to the original species description  were isolated from the sediment and washed twice in sterilized seawater.
because of the highly distinctive morphology of c. aureus and the precautions taken, the possibility of contamination is exceedingly low.
genomic dna was extracted from the cells using masterpure complete dna and rna purification kit .
the polymerase chain reaction  was performed using a total volume of  <dig> μl and the pure taq ready-to-go pcr beads kit .
nearly the entire ssu rrna gene was amplified from genomic dna using eukaryotic universal primers .
the pcr protocol had an initial denaturation stage at 95°c for  <dig> min;  <dig> cycles involving 94°c for  <dig> s , 55°c for  <dig> s , and 72°c for  <dig>  min ; and final extension at 72°c for  <dig> min.
the amplified dna fragments were purified from agarose gels using ultraclean  <dig> dna purification kit , and then cloned into the topo ta cloning kit .
the c. aureus sequence was deposited in ddbj/embl/genbank under the accession number eu <dig>
the ssu rrna sequence of c. aureus was visually aligned with taxa representing all of the major groups of eukaryotes, forming  a 38-taxon alignment with ambiguously aligned regions excluded .
in order to more comprehensively evaluate the phylogenetic position of c. aureus within the euglenozoa, we analyzed three additional datasets:  a 35-taxon alignment of euglenozoan sequences and ten relatively short environmental sequences ;  a 29-taxon alignment of euglenozoan sequences including three fast-evolving euglenid sequences – namely astasia torta , menoidium bibacillatum  and ploeotia costata  – and excluding the short environmental sequences ; and  a 25-taxon alignment of euglenozoan sequences excluding both the short environmental sequences and the fastest-evolving euglenid sequences .
the highly divergent sequences from phagotrophic euglenids produced a large number of ambiguously aligned regions in the 35-taxon and 29-taxon alignments; accordingly, these regions were excluded from our analyses.
phyml  <cit>  was used to analyze all four datasets  with maximum-likelihood  using a general-time reversible  model of base substitutions  <cit>  that incorporated invariable sites and a discrete gamma distribution  .
the gtr model was selected using the program mraic  <dig> .
<dig> with phyml http://www.abc.se/~nylander/mraic/mraic.html.
model parameters were estimated from each of the original datasets.
ml bootstrap analysis was conducted with the same settings described above .
the four alignments were also analyzed with bayesian methods using the mrbayes program  <cit> .
the program was set to operate with a gamma distribution and four monte-carlo-markov chains  starting from a random tree.
a total of  <dig> , <dig> generations were calculated with trees sampled every  <dig> generations and with a prior burn-in of  <dig>  generations .
a majority rule consensus tree was constructed from  <dig>  post-burn-in trees.
posterior probabilities correspond to the frequency at which a given node was found in the post-burn-in trees.
independent bayesian runs on each alignment yielded the same results.
archiving
a digital archive of this paper is available from pubmed central and print copies are available from libraries in the following five museums: natural history museum library , american museum of natural history , muséum national d'histoire naturelle , russian academy of sciences  and academia sinica .
RESULTS
general morphology
calkinsia aureus ranged from  <dig> – <dig>  μm long  and from  <dig> – <dig>  μm wide .
the oval-shaped cells were distinctively orange in color, dorsoventrally compressed, and possessed a tapered tail that was about  <dig> μm long .
two heterodynamic flagella were inserted within a subapical depression at the anterior end of the cell.
the longer anterior flagellum was about twice the length of the cell and was held straight forward during gliding.
the shorter posterior flagellum was half the length of the cell and was usually positioned within a ventral groove.
colorless rod-shaped epibiotic bacteria were oriented along the longitudinal axis of the cell .
the posterior half of the cell usually contained an accumulation of spherical food bodies, some of which contained diatom frustules .
cyst formation and sexual reproduction were not observed.
asexual reproduction was achieved by cell division along the longitudinal axis of the cell.
following the replication of the flagellar apparatus, a cleavage furrow formed at the anterior end of the cell and advanced toward the posterior end of the cell .
cell surface and extracellular matrix
the longitudinally arranged, epibiotic bacteria consisted of only one rod-shaped morphotype  that collectively formed a dense coat over the entire surface of the host cell .
at least  <dig> epibiotic bacteria were observed in transverse sections through one cell of c. aureus .
the cell surface beneath the epibiotic bacteria consisted of seven main layers :  a glycocalyx,  a highly organized extracellular matrix,  the host cell membrane,  an array of parallel microtubules,  a double-layered lamella,  superficially arranged mitochondrion-derived organelles and  cisternae of endoplasmic reticulum  .
the extracellular matrix surrounded the entire cell except for the inside lining of the vestibulum, which leads to the flagellar pocket and feeding pockets .
the portion of the extracellular matrix positioned just inside the opening of the vestibulum lacked epibiotic bacteria and consisted of fine hair-like structures, or somatonemes .
the extracellular matrix beneath the epibiotic bacteria was coated with a thin glycocalyx .
the extracellular matrix itself was bright orange, approximately  <dig> nm thick and perforated with hollow tubes that joined the plasma membrane of the host with the glycocalyx beneath the epibiotic bacteria .
an array of evenly spaced microtubules was positioned immediately beneath the plasma membrane of the host .
these microtubules were derived from the dorsal lamina  of the flagellar apparatus .
each microtubule was connected to neighboring microtubules with electron dense "arms" .
a double-layered lamella was positioned between the layer of microtubules and a deeper layer of mitochondrion-derived organelles .
the mitochondrion-derived organelles were discoidal in shape, were bounded by two membranes and lacked mitochondrial cristae or inclusions such as kinetoplasts .
moreover, we did not observe any evidence of euglenid-like pellicle features, such as the presence of s-shape proteinaceous strips or discontinuities in the layer of microtubules.
nucleus, vestibulum and associated pockets
an anterior nucleus was positioned near the ventral side of the cell and contained a prominent nucleolus and condensed chromosomes .
the vestibulum was positioned directly above the nucleus as this space passed from the ventral, subapical opening toward the dorsal side of the cell .
the vestibulum then extended posteriorly along the dorsal side of the cell and branched into three distinct pockets:  a novel "extrusomal pocket",  a flagellar pocket and  a feeding pocket .
a battery of longitudinally arranged extrusomes was connected to the base of the extrusomal pocket and was nested within a notch on the dorsal side of the ventral nucleus .
each extrusome was about  <dig> nm in diam.
.
the battery of extrusomes was indistinguishable from the feeding rods of euglenids when viewed with the light microscope, and discharged as a single unit through the anterior opening .
the flagellar pocket was located on the dorsal side of the cell and contained two flagella that inserted at the bottom of the pocket .
the feeding pocket was located to the right of the flagellar pocket and extended horizontally before tapering posteriorly toward the ventral side of the cell .
flagella, transition zones and basal bodies
both flagella contained a paraxonemal rod adjacent to the axoneme, and flagellar hairs were not observed on either flagellum .
the paraxonemal rod in the dorsal flagellum  had a whorled morphology in transverse section, and the paraxonemal rod in the ventral flagellum  was constructed of a three-dimensional lattice of parallel fibers .
the entire length of the axoneme had the standard 9+ <dig> architecture of microtubules .
the central microtubules within each axoneme terminated approximately  <dig> μm above the flagellar insertion ; this  <dig> +  <dig> arrangement is recognized as the transition zone in accordance with the definition proposed by hibberd  <cit> .
the transition zone and basal bodies are further described here from the distal end toward the proximal end.
the central space within the proximal half of the transition zone contained three distinct elements: faint spokes , an outer concentric ring positioned just inside the microtubular doublets , and electron dense globules  .
each faint spoke extended from a microtubular doublet toward the center of the transition zone.
the globules were positioned at the intersections of each faint spoke and the outer concentric ring .
in more proximal points along the transition zone, nine "radial connectives" extended from each doublet toward the flagellar membrane , and an opaque core was present within the central space when observed in both longitudinal and transverse section .
the opaque core consisted of six distinct elements: nine spokes extending from each doublet , the outer concentric ring , nine electron dense globules associated with the outer concentric ring , a central electron dense hub , an inner concentric ring  and nine radial connectives extending from each doublet to the flagellar membrane  .
the radial connectives disappeared just above the distal boundary of the basal body , and the elements within the central space disappeared just below the distal boundary of the basal body .
the dorsal basal body  and ventral basal body  anchored the dorsal flagellum  and ventral flagellum , respectively.
both basal bodies were approximately  <dig>  μm long, arranged in parallel to each other, and possessed nine transitional fibers extending from each triplet towards the cell membrane .
internal cartwheel elements were present within the most proximal ends of both basal bodies .
flagellar root system
the flagellar root system is described here from the proximal boundary of the basal bodies toward the distal boundary of the basal bodies as viewed from the anterior end of the cell .
the db and the vb were joined with a connecting fiber and associated with three microtubular roots: the dorsal root , the intermediate root  and the ventral root  .
the vb, ir and vr were also associated with three fibrous roots: the right fiber , the intermediate fiber  and the left fiber  .
the dr and ir were associated with two thin laminae: the dorsal lamina  and the ir-associated lamina , respectively .
the dr originated from the dorsal side of the db and consisted of three microtubules near the proximal boundary.
both the dr and the dl extended toward the anterior side of the cell  and supported the flagellar pocket .
the dr occupied the dorsal left side of the flagellar pocket; the dl occupied the dorsal right side of the flagellar pocket and extended from the vr to the dr at the level of the transition zone .
a row of linked microtubules  originated in close association with the dl  and supported the right side of the flagellar pocket .
the dl and lmt extended from the left side of the flagellar pocket to the right side near the posterior boundary of the vestibulum .
the lmt supported the inner lining of the vestibulum, turned posteriorly along the curve formed by the ventral opening  and ultimately became the sheet of microtubules located beneath the plasma membrane of the entire cell .
the ir was positioned between the two basal bodies, originated from the right dorsal side of the vb, and consisted of four microtubules near the proximal boundary .
the left side of the ir was tightly associated with the il and two fibrous roots: the lf and the if .
the lf extended laterally and was about  <dig> nm long; the if extended to the left ventral side of the cell and was about  <dig>  μm long .
the il was associated with the left side of the ir along its entire length, and the ir and il became more closely associated as they extended anteriorly along the left side of the flagellar pocket .
the microtubules from the ir eventually merged with the left side of the lmt-dl and likely contributed to the sheet of microtubules located beneath the plasma membrane of the entire cell .
the vr originated from the ventral side of the vb and consisted of nine microtubules that were closely associated with the rf .
the rf extended toward the right-ventral side of the cell and was about  <dig> μm long .
the microtubules from the vr supported the right side of the flagellar pocket and joined the right side of the lmt and the dl .
the microtubules from the vr ultimately became one of the elements that reinforced the feeding apparatus .
feeding apparatus
the feeding apparatus was positioned on the right side of the flagellar pocket and is described here along the posterior to anterior axis.
this apparatus consisted of four main elements or spaces: a feeding pocket, a vr embedded within six electron-dense fibers, a compact "oblique striated fiber"  and a "congregated globule structure"  .
the osf was approximately  <dig>  μm long,  <dig> nm wide and  <dig> nm high and was positioned between the feeding apparatus and the right side of the flagellar pocket .
the cgs attached to the anterior side of the osf .
six rows of electron dense fibers, each containing a microtubule from the vr, passed over the top of the osf-cgs complex .
the vr and the six associated fibers reinforced the anterior-right side of the feeding pocket .
the left side of the feeding pocket was reinforced by a striated fiber that extended from the left side of the cgs .
the feeding pocket was surrounded by an accumulation of small vesicles and branched from the vestibulum toward the ventral side of the cell before turning toward the posterior end of the cell .
serial oblique sections through the feeding pocket did not demonstrate distinctive feeding vanes or rods per se; only the vr microtubules within the electron dense fibers were observed .
nonetheless, the vestibular junction  between the flagellar pocket and the feeding pocket contained a "tomentum"  <cit>  of fine hairs .
molecular phylogenetic position as inferred from ssu rdna
we determined the nearly complete sequence of the ssu rrna gene of c. aureus .
maximum likelihood  analyses of  a 38-taxon alignment including representative sequences from the major lineages of eukaryotes, robustly grouped the sequence from c. aureus with the euglenozoa  .
in order to  more comprehensively evaluate the phylogenetic position of c. aureus within the euglenozoa, we analyzed three additional datasets:  a 35-taxon alignment ,  a 29-taxon alignment , and  a 25-taxon alignment  .
tree topologies of these three ml analyses were very similar .
accordingly, the results from the analyses of the 35-taxon dataset including several short environmental sequences, was an accurate representation of all three analyses .
euglenozoan sequences clustered into five major groups with high statistical support: a kinetoplastid clade, a diplonemid clade, a bacteriovorous euglenid clade , a clade consisting of eukaryovorous and photosynthetic euglenids, and a novel clade referred to here as the "symbiontida".
the relationships among these clades  were not resolved .
additional phylogenetic analyses using alternative outgroups  recovered the same basic tree topology shown in figure 11:  calkinsia aureus is a member of a distinct euglenozoan subclade consisting of sequences derived from environmental pcr surveys, and  this clade is not convincingly affiliated with any one of the three known euglenozoan subgroups .
moreover, the sequence from c. aureus occupied the deepest position within the symbiontida, which otherwise consisted of seven environmental sequences collected from northern europe and south america .
discussion
several poorly studied flagellates, some with discoidal-shaped mitochondrial cristae, have, at one time or another, been suspected to be close relatives of euglenozoans   <cit> .
the best synapomophies for the euglenozoa are  a tripartite flagellar root system ,  heteromorphic paraxonemal rods , and  tubular extrusomes  <cit> .
the presence of these ultrastructural features in very diverse lineages of flagellates, in combination with molecular phylogenetic data, has established the identity and composition of the euglenozoa  <cit> .
calkinsia aureus was originally described as a member of the euglenida with light microscopical information  <cit> , and we demonstrate here that these flagellates possess all three ultrastructural synapomorphies for the euglenozoa.
moreover, the permanently condensed chromatin, long flagellar transition zone, longitudinal cell division and long basal bodies are also features found in many other euglenozoans  <cit> .
these morphological data were concordant with our comparative analyses of ssu rdna showing that c. aureus is robustly embedded within the euglenozoa clade .
however, c. aureus lacked traits that are specific to any of the three previously recognized euglenozoan subgroups .
the faintly striated pellicle originally attributed to c. aureus using light microscopy is, in actuality, the longitudinally arranged rod-shaped epibiotic bacteria  <cit> .
the sheet of microtubules beneath the plasma membrane in c. aureus was continuous over the entire cell, like in kinetoplastids and diplonemids, rather than interrupted by periodic discontinuities like in euglenids  <cit>  .
there was also no clear evidence of a euglenid-like feeding apparatus consisting of rods and vanes  <cit> .
accordingly, our study indicates that with present knowledge, c. aureus should not be considered a member of the euglenida or more specifically, a member of the petalomonadidae as originally classified  <cit> .
absence of mitochondria with cristae
aerobic kinetoplastids and euglenids possess well-developed discoid-shaped cristae within their mitochondria  <cit> , and diplonemids and hemistasia possess a few flat-shaped cristae within each mitochondrion  <cit> .
by contrast, both c. aureus and p. mariagerensis lack recognizable mitochondria with cristae, and instead, contain double-membrane bound organelles that are nearly identical in morphology to the well-studied hydrogenosomes described in other anoxic flagellates   <cit> .
hydrogenosomes are the descendents of mitochondria and function to produce molecular hydrogen, acetate, co <dig> and atp in anoxic environments  <cit> .
a more confident functional characterization of the mitochondrion-derived organelles in c. aureus or postgaardi will require biochemical and molecular biological assays.
a novel extracellular matrix
the plasma membrane of c. aureus was reinforced with a continuous sheet of microtubules and a double-layered lamella, which was in turn subtended by a dense array of mitochondrion-derived organelles .
this overall organization, where mitochondrion-derived organelles are located immediately beneath a sheet of surface microtubules, has also been observed in postgaardi.
however, a uniform and perforated extracellular matrix enveloped the cell surface of c. aureus, and so far as we know, the organization of this cell covering is novel not only among euglenozoans, but also among eukaryotes .
because both the epibiotic bacteria and the host cell cytoplasm were colorless , the distinctively orange color of c. aureus is clearly attributable to the chemical composition of the extracellular matrix .
moreover, the even distribution of tiny tubes within the matrix provide conduits between the host plasma membrane and the epibiotic bacteria and presumably facilitate metabolic exchanges necessary for survival in low-oxygen environments.
this interpretation is consistent with knowledge of anoxic ciliates, which also maintain an intimate physical relationship between mitochondrion-derived organelles  and epibiotic bacteria   <cit> .
flagellar apparatus
the flagella of most euglenids and kinetoplastids have non-tubular mastigonemes  that, among other functions, facilitate gliding motility  <cit> ; however, these structures are absent in c. aureus, p. mariagerensis and diplonemids.
instead, a tomentum of fine hairs are present at the crest of the feeding pocket in c. aureus that are similar to those described in the phototrophic euglenid colacium  <cit> , the phagotrophic euglenid peranema  <cit> , and the kinetoplastid cryptobia  <cit> .
although hairs associated with the feeding pocket have not been routinely observed in other members of the euglenozoa, the presence of these hairs in distantly related lineages indicate that this feature might represent an ancestral state for the euglenozoa as a whole.
the structure of the flagellar transition zone is variable among kinetoplastids and euglenids, particularly in regard to the presence/absence of peripheral elements and transitional plates.
kinetoplastids and diplonemids possess distal and proximal transitional plates and a hollow transition zone  <cit> , while euglenids only possess the proximal transitional plate.
although the transition zone of most euglenids is also hollow, the transition zone in some euglenids, such as entosiphon applanatum and notosolenus mediocanellata, has been shown to be electron dense.
however, the detailed structure of these transition zones still remains to be characterized in detail  <cit> .
the central area of the transition zone in c. aureus is also electron dense and contains a complex system of elements that have never been observed in any other euglenozoan so far .
characterization of the flagellar transition zone in postgaardi might demonstrate several homologous elements that would help to further establish a close relationship between this lineage and c. aureus.
nonetheless, diplonema ambulator, rhynchopus euleeides, r. coscinodiscivorus and c. aureus all have fibers that extend from each microtubular doublet to the flagellar membrane; these fibers have been called "transitional fibers"  <cit> .
"transitional fibers" has also been used to describe fibers that extended from each microtubular triplet of a basal body to the flagellar membrane, which is potentially confusing  <cit> .
nonetheless, the "radial connectives" extending from the doublets in the transition zone of c. aureus are nearly identical, and likely homologous, to the 'transitional fibers' extending from the doublets in diplonemids, such as d. ambulator.
feeding apparatus
each of the euglenozoan subgroups contains members with an elaborate feeding apparatus  <cit> .
most phagotrophic euglenids, for instance, have a distinctive feeding apparatus consisting of 4– <dig> central vanes and 2– <dig> supporting rods  <cit> .
some bacteriovorous euglenids , however, possess a much simpler feeding apparatus that is very similar to the mtr feeding pockets found in many kinetoplastids   <cit> .
the microtubules that support the rods in phagotrophic euglenids and the mtr pockets in bacteriovorous euglenozoans originate from the ventral root of the ventral basal body.
similarly, the feeding pocket in c. aureus was also supported by microtubules that originated from the ventral root and is almost certainly homologous to the mtr pockets or rods found in other euglenozoans, including postgaardi  <cit> .
moreover, the compact "oblique striated fiber"  that reinforces the feeding pocket in c. aureus was similar to the amorphous matrix found in some euglenid feeding rods and might represent a vestige of a more elaborate ancestral state.
however, this inference will require improved understanding of the morphological diversity and phylogeny of other euglenozoans that are more closely related to c. aureus.
a novel extrusomal pocket
although tubular extrusomes are not widespread within the euglenozoa, several members from each main subgroup possess them, such as the euglenid entosiphon  <cit> ; the kinetoplastids rhynchobodo  <cit> , hemistasia  <cit> , and phylomitus  <cit> ; the diplonemid diplonema nigricans  <cit> ; and postgaardi mariagerensis  <cit> .
calkinsia aureus not only had tubular extrusomes like the lineages listed above, but they were clustered together much like the single battery of tubular extrusomes found in hemistasia  <cit> .
by contrast, postgaardi and rhynchobodo possess several smaller batteries of tubular extrusomes that are dispersed throughout the cytoplasm  <cit> .
the battery of tubular extrusomes in c. aureus was anchored to a novel extrusomal pocket that branched off of the vestibulum separately from the feeding apparatus and the flagellar apparatus .
this battery of extrusomes was often discharged as a single unit from the extrusomal pocket and through the anterior opening .
the functional significance of this process is unclear.
the phagotrophic euglenid dinema sulcatum also contains a flagellar pocket and reportedly has two additional pockets:  a "normal" feeding apparatus consisting of supportive rods and vanes and  an "extra" pocket consisting of mtr-like microtubules  <cit> .
one previously proposed hypothesis for the presence of two feeding pockets in d. sulcatum involves the following inferences: the "extra" pocket is a remnant of the mtr feeding pocket present in the ancestral euglenozoan and the rod-and-vane based feeding apparatus represents a duplicated, and greatly embellished, mtr pocket that arose within a derived lineage of phagotrophic euglenids  <cit> .
this hypothesis is consistent with comparative morphological data that indicates other euglenid cytoskeletal components also evolved by duplication, such as the total number of pellicle strips around the cell periphery  <cit> .
nonetheless, the extrusomal pocket in c. aureus was supported by the lmt  rather than microtubules from the ventral root, which support both mtr pockets and rod-and-vane based feeding apparatuses in euglenozoans.
therefore, the extrusomal pocket in c. aureus appears to be novel and does not seem to be homologous to any type of feeding apparatus reported so far .
euglenozoans with epibiotic bacteria
postgaardi mariagerensis  <cit> , euglena helicoideus  <cit> , dylakosoma pelophilum  <cit> , c. aureus  <cit>  and five unidentified euglenozoans from low oxygen environments  <cit>  have been reported to possess epibiotic bacteria on the cell surface.
the epibiotic bacteria on d. pelophilum are spherical, and those on the other taxa are rod-shaped and densely packed on the cell surface.
only one of the five unidentified euglenozoans, namely "morphotype c" from monterey bay, was studied with both sem and tem  <cit> .
the rod-shape epibiotic bacteria on these cells were not associated with a superficial distribution of mitochondrion-derived organelles  beneath the host plasma membrane.
nonetheless, morphotype c was clearly a euglenid, because the flagella contained paraxonemal rods, the feeding apparatus consisted of rods and vanes, and thin proteinaceous strips supported the cell surface.
by contrast, the combination of ultrastructural features in c. aureus and p. mariagerensis make these lineages difficult to place within the euglenozoa.
both lineages lack evidence of pellicle strips or kinetoplasts and possess paraxonemal rods, tubular extrusomes, mitochondrion-derived organelles beneath the plasma membrane, and condensed chromatin.
detailed comparisons of the feeding apparatus in c. aureus, p. mariagerensis, and other anoxic euglenozoans should help better establish their phylogenetic relationships with each other; however, except for c. aureus, this information is currently lacking for nearly all of these lineages, including p. mariagerensis.
molecular phylogenetic framework for euglenozoans in low-oxygen environments
the morphology of c. aureus  was completely concordant with the molecular phylogenetic data in so far as strongly placing c. aureus within the euglenozoa, but not with any of the three previously recognized subclades.
figure  <dig> shows the phylogenetic position of c. aureus within the euglenozoa, which consisted of five main clades.
although petalomonas and notosolenus branched together as a separate clade, morphological evidence strongly supports their inclusion within the euglenida.
therefore, the molecular phylogenetic data coupled with the morphological data allows us to recognize four clades of euglenozoans: the euglenida, the kinetoplastida, the diplonemida and a novel clade of anoxic euglenozoans, hereby named the symbiontida.
the symbiontida includes several environmental sequences that were originally designated either as diplonemid sequences   <cit> , as uncultured euglenozoan sequences   <cit>  or as "possible early branching eukaryotes"   <cit> .
some of the environmental sequences within the symbiontida were already suspected to represent either a novel sister clade to the euglenozoa or novel subclade of euglenozoans  <cit> .
nonetheless, we have demonstrated that the symbiontida contains several more environmental sequences collected from different low-oxygen environments and also c. aureus, which provides an organismal anchor  for this clade.
we should also note that some environmental sequences from mid-atlantic hydrothermal vent environments in the "lost city", namely lc <dig> 5ep  <dig>  lc <dig> 5ep  <dig>  and lc <dig> 5ep  <dig>  grouped strongly with the diplonemid clade and not with the symbiotida  <cit> .
moreover, the lack of phylogenetic signal and perhaps also long-branch-attraction were the likely reasons for why the relatively fast-evolving sequences from notosolenus and petalomonas did not cluster strongly with the euglenid clade in our analyses of the dataset containing the shortest sequences .
our analysis of the dataset including only the longest sequences, by contrast, clustered notosolemus and petalomonas with all other euglenids, albeit without strong statistical support   <cit> .
the symbiontida: a novel subclade of the euglenozoa
before c. aureus had been studied at the ultrastructural and molecular phylogenetic levels, one author classified this lineage with p. mariagerensis within the taxon "postgaardea" on the basis of microaerophily  <cit> .
although our characterization of c. aureus has demonstrated epibiotic bacteria and mitochondrion-derived organelles like those described in p. mariagerensis, the presence of these characters in both lineages does not necessarily reflect homology.
independently derived physical relationships between epibiotic bacteria and mitochondrion-derived organelles have been found in many different lineages of anoxic microeukaryotes, such as ciliates, oxymonads, parabasalids, heteroloboseans and euglenozoans  <cit> .
moreover, the presence of tubular extrusomes in both c. aureus and p. mariagerensis could be a symplesiomorphic state inherited from a very distant euglenozoan ancestor.
nonetheless, our phylogenetic analyses demonstrate that c. aureus is a member of a newly recognized clade of anoxic euglenozoans consisting mainly of environmental sequences.
the absence of molecular phylogenetic data and conclusive ultrastructural data from postgaardi precludes us from determining whether this lineage is also a member of the clade of microaerophiles.
until these data are reported and the phylogenetic position of postgaardi is demonstrated more rigorously, we concur with a previous taxonomic treatment for postgaardi that recognizes this lineage as incertae sedis within the euglenozoa  <cit> .
as such, we conclude that it is premature to recognize the taxon postgaardea and view it as a synonym for p. mariagerensis.
in light of the previous discussion, we propose the name "symbiontida" for the clade of microaerobic or anaerobic euglenozoans consisting of the most recent ancestor of c. aureus that also possessed rod-shaped epibiotic bacteria, reduced or absent mitochondrial cristae, tubular extrusomes and a nucleus with permanently condensed chromatin.
this novel subclade of euglenozoans is recognized on the basis of ssu rdna-based phylogenetic data of lineages from several different low-oxygen environments.
although the ultrastructural characteristics listed above are expected to be present in most, if not all, members of the symbiontida , this remains to be demonstrated with improved knowledge of euglenozoan diversity from both ultrastructural and molecular phylogenetic perspectives.
phylogenetic  diagnosis
euglenozoa cavalier-smith 1981
symbiontida taxon nov. yubuki, edgcomb, bernhard & leander, 2009
apomorphy
rod-shaped epibiotic bacteria above superficial layer of mitochondrion-derived organelles with reduced or absent cristae, homologous to the organization in calkinsia aureus, the type species .
extended diagnosis of the type species
calkinsia aureus lackey,  <dig>  emend., yubuki, edgcomb, bernhard & leander, 2009
paraxonemal rods present in flagella; kinetoplast dna and pellicle strips absent; long complex transitional zone between the basal bodies and the axonemes.
rod-shaped epibiotic bacteria on perforated orange extracellular matrix.
cell with a large nucleus on the anterior ventral side and a battery of tubular extrusomes linked to an extrusomal pocket located adjacent to the nucleus.
feeding apparatus supported by both fibrous structures and microtubules that are derived from ventral root .
small subunit ribosomal rna gene sequence  distinguishes calkinsia aureus from all other symbiontid species.
CONCLUSIONS
molecular phylogenies inferred from ssu rdna demonstrate that c. aureus is closely related to several marine environmental sequences collected from low-oxygen environments, forming a novel subgroup within the euglenozoa, referred to here as the "symbiontida".
improved understanding of these flagellates is necessary for further demonstrating the cellular identity of the symbiontida and for reconstructing the evolutionary radiation of the euglenozoan lineage.
in this study, we characterized the detailed ultrastructure of c. aureus and demonstrated all of the euglenozoan synapomorphies  and several cellular innovations associated with symbiotic interactions with epibiotic bacteria .
we also demonstrated novel ultrastructural systems found in this species, such as the extrusomal pocket.
environmental sequencing surveys from different low-oxygen environments around the world suggest that many symbiontid lineages have yet to be discovered and characterized.
continued exploration into the overall diversity of this group should contribute significantly to our understanding of eukaryotic evolution, especially in low-oxygen environments.
abbreviations
af: anterior flagellum; b: epibiotic bacteria; ba: bacteriotroph; cgs: congregated globule structure; db: dorsal basal body; df: dorsal flagellum; dl: dorsal lamina; dr: dorsal root; e: extrusome; ep: extrusomal pocket; eu: eukaryotroph; ex: extracellular matrix; g: golgi body; gl: glycocalyx; if: intermediate fiber; il: ir-associated lamina; ir: intermediate root; lf: left fiber; lm: light microscope; lmt: linked microtubules; mtd: mitochondrion-derived organelle; n: nucleus; nu: nucleolus; os: osmotroph; osf: oblique striated fibrous structure; pf: posterior flagellum; ph: phototroph; rf: right fiber; s: somatonema; sbb: santa barbara basin; scb: sodium cacodylate buffer; sem: scanning electron microscope; vb: ventral basal body; t: tomentum; tem: transmission electron microscope; vf: ventral flagellum; vr: ventral root.
authors' contributions
ny carried out all of the lm, sem, tem and molecular phylogenetic work, wrote the first draft of the paper and participated in the collection of sediment samples from the sbb.
vpe and jmb, the chief scientist, coordinated and funded the research cruise to the sbb.
bsl funded and supervised the collection and interpretation of the ultrastructural and molecular phylogenetic data and contributed to writing the paper.
all authors have read, edited, and approved the final manuscript.
supplementary material
additional file 1
maximum likelihood  analysis of  <dig> taxa focusing on the position of calkinsia aureus within the euglenozoa clade.
two jakobids, andalucia incarcerata and a. godoyi, are used as outgroups in this analysis.
the short environmental sequences are excluded from the dataset used in figure  <dig> and fast-evolve euglenids sequences, ploeotia, menoidium and astasia, are included.
ml bootstrap values greater than 50% are shown.
thick branches indicate bayesian posterior probabilities over  <dig> .
ba, bacteriotroph; eu, eukaryotroph; os, osmotroph; ph, phototroph.
genbank accession numbers of the sequences analyzed are shown in parentheses.
click here for file
additional file 2
maximum likelihood  analysis of  <dig> taxa focusing on the position of calkinsia aureus within the euglenozoa clade.
two jakobids, andalucia incarcerata and a. godoyi, are used as outgroups in this analysis.
the short environmental sequences are removed from the dataset used in figure  <dig> and fast-evolve euglenids sequences, dinema, ploeotia, menoidium and astasia, are excluded.
ml bootstrap values greater than 50% are shown.
thick branches indicate bayesian posterior probabilities over  <dig> .
ba, bacteriotroph; eu, eukaryotroph; ph, phototroph.
genbank accession numbers of the sequences analyzed are shown in parentheses.
click here for file
acknowledgements
this work was supported by grants to bsl from the tula foundation , the national science and engineering research council of canada  and the canadian institute for advanced research, program in integrated microbial biodiversity.
funding for the collection of sediments and participation of vpe and jmb in this research was provided by the us national science foundation grant mcb- <dig>  we also acknowledge the constructive feedback from four anonymous reviewers.

 the euglenozoa is a clade of eukaryotic microorganisms with very diverse lifestyles and that tentatively falls within one of six emerging supergroups of eukaryotes, namely the "excavata". most euglenozoans cluster within three major subgroups that have been established with both molecular phylogenetic analyses and combination of ultrastructural characteristics: the kinetoplastida, the euglenida and the diplonemida. kinetoplastids possess mitochondria with a uniquely structured genome, called "kinetoplast" dna, and the group includes both free-living phagotrophic lineages and parasitic lineages. euglenids possess a cytoskeleton, or "pellicle", consisting of overlapping proteinaceous strips that are arranged either longitudinally or helically, and the group includes bacteriovorous lineages, eukaryovorous lineages, osmotrophic lineages and photosynthetic lineages. the mitochondria of kinetoplastids and euglenids possess cristae that are distinctively discoidal in shape. by contrast, diplonemids consist of only two genera, diplonema and rhynchopus, with sack-shaped cells, short flagella and flattened mitochondrial cristae and without kinetoplast dna, pellicle strips, and paraxonemal rods. ultrastructural studies have also demonstrated lineages of euglenozoans that do not fall neatly within any of the three established subgroups, such as postgaardi mariagerensis, which inhabits low oxygen environments and is covered with epibiotic bacteria. currently, p. mariagerensis is grouped together with another poorly understood anoxic flagellate, namely calkinsia aureus, as incertae sedis within the euglenozoa; although molecular data is unavailable for both species, one author has chosen to classify them within a taxon called the "postgaardea". c. aureus was originally collected from anoxic sediments near woods hole, ma and described with only line drawings as a member of the euglenid family petalomonidae; this conclusion was based on the appearance of a rigid cell containing strip-like surface striations. however, c. aureus was subsequently collected from low-oxygen sediments in the santa barbara basin, ca and partially studied with light and scanning electron microscopy. these studies demonstrated that like p. mariagerensis, c. aureus was covered with the rod-shape epibiotic bacteria, rather than pellicle strips per se. the ultrastructure and molecular phylogenetic position of c. aureus is currently unknown. these data are expected to help establish robust inferences about the overall diversity of euglenozoans and the ultrastructure of prokaryote-eukaryote symbioses within the group and beyond. the main goals of this study were to characterize the ultrastructure and molecular phylogenetic position of c. aureus using small subunit rdna sequences and transmission electron microscopy of serially sectioned cells. our results demonstrated that c. aureus is the first member of a novel group of anoxic euglenozoans – referred to here as the "symbiontida" – to be characterized at both the molecular and ultrastructural levels. a companion study centered on the molecular identity and detailed ultrastructure of the epibiotic bacteria on c. aureus is currently underway. methods collection of organisms calkinsia aureus was collected using a soutar box corer or mc- multi corer from the sea floor sediment of the santa barbara basin, california, usa in september of and june of sediment core samples were collected on the r/v robert gordon sproul. some sediment samples were immediately fixed for transmission electron microscopy with an equal volume of 4% glutaraldehyde in m sodium cacodylate buffer and stored at 4°c. the remaining sediment samples were stored in ml plastic tubes at 4°c and subsequently processed for light microscopy, scanning electron microscopy and dna extraction. light and electron microscopy light micrographs of over living cells were taken using a zeiss axioplan imaging microscope and a leica dc digital chilled ccd camera. cells of c. aureus were prepared for sem by mixing an equal volume of fixative solution containing 4% glutaraldehyde in m scb at room temperature. the fixed cells were mounted on polycarbonate millipore filters or glass plates coated with poly-l-lysine at room temperature for hr. the cells were rinsed with m scb and fixed in 1% osmium tetroxide for min. the osmium-fixed cells were then rinsed with m scb and dehydrated with a graded ethanol series from 30% to absolute ethanol before being critical point dried with co using a tousimis critical point dryer. the dried cells were then coated with gold using a cressington 208hr high resolution sputter coater, and observed with a hitachi s- field emission scanning electron microscope. cells of c. aureus prepared for tem were kept in fixative solution for two months before being individually isolated from the surrounding sediment in the sample. isolated cells were rinsed with m scb three times and then fixed in 1% osmium tetroxide in m scb at room temperature for hr before being dehydrated through a graded series of ethanol and 100% acetone. the dehydrated cells were then infiltrated with acetone-epon resin mixtures and 100% resin. individual cells were flat embedded and serial sectioned in different orientations. ultra-thin serial sections were collected on copper, formvar-coated slot grids and stained with 2% uranyl acetate and lead citrate before being observed using a hitachi h electron microscope. a total of micrographs from different cells were observed. two different media were used in an attempt to culture c. aureus: 5% of tygm- and 5% of modified pynfh medium, diluted in anoxic and axenic seawater at 4°c. however, the cells did not grow in either medium. dna extraction, pcr amplification, alignment and phylogenetic analysis twenty individual cells of c. aureus that corresponded to the original species description were isolated from the sediment and washed twice in sterilized seawater. because of the highly distinctive morphology of c. aureus and the precautions taken, the possibility of contamination is exceedingly low. genomic dna was extracted from the cells using masterpure complete dna and rna purification kit. the polymerase chain reaction was performed using a total volume of μl and the pure taq ready-to-go pcr beads kit. nearly the entire ssu rrna gene was amplified from genomic dna using eukaryotic universal primers. the pcr protocol had an initial denaturation stage at 95°c for min; cycles involving 94°c for s, 55°c for s, and 72°c for min; and final extension at 72°c for min. the amplified dna fragments were purified from agarose gels using ultraclean dna purification kit, and then cloned into the topo ta cloning kit. the c. aureus sequence was deposited in ddbj/embl/genbank under the accession number eu the ssu rrna sequence of c. aureus was visually aligned with taxa representing all of the major groups of eukaryotes, forming a 38-taxon alignment with ambiguously aligned regions excluded. in order to more comprehensively evaluate the phylogenetic position of c. aureus within the euglenozoa, we analyzed three additional datasets: a 35-taxon alignment of euglenozoan sequences and ten relatively short environmental sequences; a 29-taxon alignment of euglenozoan sequences including three fast-evolving euglenid sequences – namely astasia torta, menoidium bibacillatum and ploeotia costata – and excluding the short environmental sequences; and a 25-taxon alignment of euglenozoan sequences excluding both the short environmental sequences and the fastest-evolving euglenid sequences. the highly divergent sequences from phagotrophic euglenids produced a large number of ambiguously aligned regions in the 35-taxon and 29-taxon alignments; accordingly, these regions were excluded from our analyses. phyml was used to analyze all four datasets with maximum-likelihood using a general-time reversible model of base substitutions that incorporated invariable sites and a discrete gamma distribution. the gtr model was selected using the program mraic. with phyml http://www.abc.se/~nylander/mraic/mraic.html. model parameters were estimated from each of the original datasets. ml bootstrap analysis was conducted with the same settings described above. the four alignments were also analyzed with bayesian methods using the mrbayes program. the program was set to operate with a gamma distribution and four monte-carlo-markov chains starting from a random tree. a total of, generations were calculated with trees sampled every generations and with a prior burn-in of generations. a majority rule consensus tree was constructed from post-burn-in trees. posterior probabilities correspond to the frequency at which a given node was found in the post-burn-in trees. independent bayesian runs on each alignment yielded the same results. archiving a digital archive of this paper is available from pubmed central and print copies are available from libraries in the following five museums: natural history museum library, american museum of natural history, muséum national d'histoire naturelle, russian academy of sciences and academia sinica.  general morphology calkinsia aureus ranged from – μm long and from – μm wide. the oval-shaped cells were distinctively orange in color, dorsoventrally compressed, and possessed a tapered tail that was about μm long. two heterodynamic flagella were inserted within a subapical depression at the anterior end of the cell. the longer anterior flagellum was about twice the length of the cell and was held straight forward during gliding. the shorter posterior flagellum was half the length of the cell and was usually positioned within a ventral groove. colorless rod-shaped epibiotic bacteria were oriented along the longitudinal axis of the cell. the posterior half of the cell usually contained an accumulation of spherical food bodies, some of which contained diatom frustules. cyst formation and sexual reproduction were not observed. asexual reproduction was achieved by cell division along the longitudinal axis of the cell. following the replication of the flagellar apparatus, a cleavage furrow formed at the anterior end of the cell and advanced toward the posterior end of the cell. cell surface and extracellular matrix the longitudinally arranged, epibiotic bacteria consisted of only one rod-shaped morphotype that collectively formed a dense coat over the entire surface of the host cell. at least epibiotic bacteria were observed in transverse sections through one cell of c. aureus. the cell surface beneath the epibiotic bacteria consisted of seven main layers: a glycocalyx, a highly organized extracellular matrix, the host cell membrane, an array of parallel microtubules, a double-layered lamella, superficially arranged mitochondrion-derived organelles and cisternae of endoplasmic reticulum. the extracellular matrix surrounded the entire cell except for the inside lining of the vestibulum, which leads to the flagellar pocket and feeding pockets. the portion of the extracellular matrix positioned just inside the opening of the vestibulum lacked epibiotic bacteria and consisted of fine hair-like structures, or somatonemes. the extracellular matrix beneath the epibiotic bacteria was coated with a thin glycocalyx. the extracellular matrix itself was bright orange, approximately nm thick and perforated with hollow tubes that joined the plasma membrane of the host with the glycocalyx beneath the epibiotic bacteria. an array of evenly spaced microtubules was positioned immediately beneath the plasma membrane of the host. these microtubules were derived from the dorsal lamina of the flagellar apparatus. each microtubule was connected to neighboring microtubules with electron dense "arms". a double-layered lamella was positioned between the layer of microtubules and a deeper layer of mitochondrion-derived organelles. the mitochondrion-derived organelles were discoidal in shape, were bounded by two membranes and lacked mitochondrial cristae or inclusions such as kinetoplasts. moreover, we did not observe any evidence of euglenid-like pellicle features, such as the presence of s-shape proteinaceous strips or discontinuities in the layer of microtubules. nucleus, vestibulum and associated pockets an anterior nucleus was positioned near the ventral side of the cell and contained a prominent nucleolus and condensed chromosomes. the vestibulum was positioned directly above the nucleus as this space passed from the ventral, subapical opening toward the dorsal side of the cell. the vestibulum then extended posteriorly along the dorsal side of the cell and branched into three distinct pockets: a novel "extrusomal pocket", a flagellar pocket and a feeding pocket. a battery of longitudinally arranged extrusomes was connected to the base of the extrusomal pocket and was nested within a notch on the dorsal side of the ventral nucleus. each extrusome was about nm in diam.. the battery of extrusomes was indistinguishable from the feeding rods of euglenids when viewed with the light microscope, and discharged as a single unit through the anterior opening. the flagellar pocket was located on the dorsal side of the cell and contained two flagella that inserted at the bottom of the pocket. the feeding pocket was located to the right of the flagellar pocket and extended horizontally before tapering posteriorly toward the ventral side of the cell. flagella, transition zones and basal bodies both flagella contained a paraxonemal rod adjacent to the axoneme, and flagellar hairs were not observed on either flagellum. the paraxonemal rod in the dorsal flagellum had a whorled morphology in transverse section, and the paraxonemal rod in the ventral flagellum was constructed of a three-dimensional lattice of parallel fibers. the entire length of the axoneme had the standard 9+ architecture of microtubules. the central microtubules within each axoneme terminated approximately μm above the flagellar insertion; this + arrangement is recognized as the transition zone in accordance with the definition proposed by hibberd. the transition zone and basal bodies are further described here from the distal end toward the proximal end. the central space within the proximal half of the transition zone contained three distinct elements: faint spokes, an outer concentric ring positioned just inside the microtubular doublets, and electron dense globules. each faint spoke extended from a microtubular doublet toward the center of the transition zone. the globules were positioned at the intersections of each faint spoke and the outer concentric ring. in more proximal points along the transition zone, nine "radial connectives" extended from each doublet toward the flagellar membrane, and an opaque core was present within the central space when observed in both longitudinal and transverse section. the opaque core consisted of six distinct elements: nine spokes extending from each doublet, the outer concentric ring, nine electron dense globules associated with the outer concentric ring, a central electron dense hub, an inner concentric ring and nine radial connectives extending from each doublet to the flagellar membrane. the radial connectives disappeared just above the distal boundary of the basal body, and the elements within the central space disappeared just below the distal boundary of the basal body. the dorsal basal body and ventral basal body anchored the dorsal flagellum and ventral flagellum, respectively. both basal bodies were approximately μm long, arranged in parallel to each other, and possessed nine transitional fibers extending from each triplet towards the cell membrane. internal cartwheel elements were present within the most proximal ends of both basal bodies. flagellar root system the flagellar root system is described here from the proximal boundary of the basal bodies toward the distal boundary of the basal bodies as viewed from the anterior end of the cell. the db and the vb were joined with a connecting fiber and associated with three microtubular roots: the dorsal root, the intermediate root and the ventral root. the vb, ir and vr were also associated with three fibrous roots: the right fiber, the intermediate fiber and the left fiber. the dr and ir were associated with two thin laminae: the dorsal lamina and the ir-associated lamina, respectively. the dr originated from the dorsal side of the db and consisted of three microtubules near the proximal boundary. both the dr and the dl extended toward the anterior side of the cell and supported the flagellar pocket. the dr occupied the dorsal left side of the flagellar pocket; the dl occupied the dorsal right side of the flagellar pocket and extended from the vr to the dr at the level of the transition zone. a row of linked microtubules originated in close association with the dl and supported the right side of the flagellar pocket. the dl and lmt extended from the left side of the flagellar pocket to the right side near the posterior boundary of the vestibulum. the lmt supported the inner lining of the vestibulum, turned posteriorly along the curve formed by the ventral opening and ultimately became the sheet of microtubules located beneath the plasma membrane of the entire cell. the ir was positioned between the two basal bodies, originated from the right dorsal side of the vb, and consisted of four microtubules near the proximal boundary. the left side of the ir was tightly associated with the il and two fibrous roots: the lf and the if. the lf extended laterally and was about nm long; the if extended to the left ventral side of the cell and was about μm long. the il was associated with the left side of the ir along its entire length, and the ir and il became more closely associated as they extended anteriorly along the left side of the flagellar pocket. the microtubules from the ir eventually merged with the left side of the lmt-dl and likely contributed to the sheet of microtubules located beneath the plasma membrane of the entire cell. the vr originated from the ventral side of the vb and consisted of nine microtubules that were closely associated with the rf. the rf extended toward the right-ventral side of the cell and was about μm long. the microtubules from the vr supported the right side of the flagellar pocket and joined the right side of the lmt and the dl. the microtubules from the vr ultimately became one of the elements that reinforced the feeding apparatus. feeding apparatus the feeding apparatus was positioned on the right side of the flagellar pocket and is described here along the posterior to anterior axis. this apparatus consisted of four main elements or spaces: a feeding pocket, a vr embedded within six electron-dense fibers, a compact "oblique striated fiber" and a "congregated globule structure". the osf was approximately μm long, nm wide and nm high and was positioned between the feeding apparatus and the right side of the flagellar pocket. the cgs attached to the anterior side of the osf. six rows of electron dense fibers, each containing a microtubule from the vr, passed over the top of the osf-cgs complex. the vr and the six associated fibers reinforced the anterior-right side of the feeding pocket. the left side of the feeding pocket was reinforced by a striated fiber that extended from the left side of the cgs. the feeding pocket was surrounded by an accumulation of small vesicles and branched from the vestibulum toward the ventral side of the cell before turning toward the posterior end of the cell. serial oblique sections through the feeding pocket did not demonstrate distinctive feeding vanes or rods per se; only the vr microtubules within the electron dense fibers were observed. nonetheless, the vestibular junction between the flagellar pocket and the feeding pocket contained a "tomentum" of fine hairs. molecular phylogenetic position as inferred from ssu rdna we determined the nearly complete sequence of the ssu rrna gene of c. aureus. maximum likelihood analyses of a 38-taxon alignment including representative sequences from the major lineages of eukaryotes, robustly grouped the sequence from c. aureus with the euglenozoa. in order to more comprehensively evaluate the phylogenetic position of c. aureus within the euglenozoa, we analyzed three additional datasets: a 35-taxon alignment, a 29-taxon alignment, and a 25-taxon alignment. tree topologies of these three ml analyses were very similar. accordingly, the results from the analyses of the 35-taxon dataset including several short environmental sequences, was an accurate representation of all three analyses. euglenozoan sequences clustered into five major groups with high statistical support: a kinetoplastid clade, a diplonemid clade, a bacteriovorous euglenid clade, a clade consisting of eukaryovorous and photosynthetic euglenids, and a novel clade referred to here as the "symbiontida". the relationships among these clades were not resolved. additional phylogenetic analyses using alternative outgroups recovered the same basic tree topology shown in figure 11: calkinsia aureus is a member of a distinct euglenozoan subclade consisting of sequences derived from environmental pcr surveys, and this clade is not convincingly affiliated with any one of the three known euglenozoan subgroups. moreover, the sequence from c. aureus occupied the deepest position within the symbiontida, which otherwise consisted of seven environmental sequences collected from northern europe and south america. discussion several poorly studied flagellates, some with discoidal-shaped mitochondrial cristae, have, at one time or another, been suspected to be close relatives of euglenozoans. the best synapomophies for the euglenozoa are a tripartite flagellar root system, heteromorphic paraxonemal rods, and tubular extrusomes. the presence of these ultrastructural features in very diverse lineages of flagellates, in combination with molecular phylogenetic data, has established the identity and composition of the euglenozoa. calkinsia aureus was originally described as a member of the euglenida with light microscopical information, and we demonstrate here that these flagellates possess all three ultrastructural synapomorphies for the euglenozoa. moreover, the permanently condensed chromatin, long flagellar transition zone, longitudinal cell division and long basal bodies are also features found in many other euglenozoans. these morphological data were concordant with our comparative analyses of ssu rdna showing that c. aureus is robustly embedded within the euglenozoa clade. however, c. aureus lacked traits that are specific to any of the three previously recognized euglenozoan subgroups. the faintly striated pellicle originally attributed to c. aureus using light microscopy is, in actuality, the longitudinally arranged rod-shaped epibiotic bacteria. the sheet of microtubules beneath the plasma membrane in c. aureus was continuous over the entire cell, like in kinetoplastids and diplonemids, rather than interrupted by periodic discontinuities like in euglenids. there was also no clear evidence of a euglenid-like feeding apparatus consisting of rods and vanes. accordingly, our study indicates that with present knowledge, c. aureus should not be considered a member of the euglenida or more specifically, a member of the petalomonadidae as originally classified. absence of mitochondria with cristae aerobic kinetoplastids and euglenids possess well-developed discoid-shaped cristae within their mitochondria, and diplonemids and hemistasia possess a few flat-shaped cristae within each mitochondrion. by contrast, both c. aureus and p. mariagerensis lack recognizable mitochondria with cristae, and instead, contain double-membrane bound organelles that are nearly identical in morphology to the well-studied hydrogenosomes described in other anoxic flagellates. hydrogenosomes are the descendents of mitochondria and function to produce molecular hydrogen, acetate, co and atp in anoxic environments. a more confident functional characterization of the mitochondrion-derived organelles in c. aureus or postgaardi will require biochemical and molecular biological assays. a novel extracellular matrix the plasma membrane of c. aureus was reinforced with a continuous sheet of microtubules and a double-layered lamella, which was in turn subtended by a dense array of mitochondrion-derived organelles. this overall organization, where mitochondrion-derived organelles are located immediately beneath a sheet of surface microtubules, has also been observed in postgaardi. however, a uniform and perforated extracellular matrix enveloped the cell surface of c. aureus, and so far as we know, the organization of this cell covering is novel not only among euglenozoans, but also among eukaryotes. because both the epibiotic bacteria and the host cell cytoplasm were colorless, the distinctively orange color of c. aureus is clearly attributable to the chemical composition of the extracellular matrix. moreover, the even distribution of tiny tubes within the matrix provide conduits between the host plasma membrane and the epibiotic bacteria and presumably facilitate metabolic exchanges necessary for survival in low-oxygen environments. this interpretation is consistent with knowledge of anoxic ciliates, which also maintain an intimate physical relationship between mitochondrion-derived organelles and epibiotic bacteria. flagellar apparatus the flagella of most euglenids and kinetoplastids have non-tubular mastigonemes that, among other functions, facilitate gliding motility; however, these structures are absent in c. aureus, p. mariagerensis and diplonemids. instead, a tomentum of fine hairs are present at the crest of the feeding pocket in c. aureus that are similar to those described in the phototrophic euglenid colacium, the phagotrophic euglenid peranema, and the kinetoplastid cryptobia. although hairs associated with the feeding pocket have not been routinely observed in other members of the euglenozoa, the presence of these hairs in distantly related lineages indicate that this feature might represent an ancestral state for the euglenozoa as a whole. the structure of the flagellar transition zone is variable among kinetoplastids and euglenids, particularly in regard to the presence/absence of peripheral elements and transitional plates. kinetoplastids and diplonemids possess distal and proximal transitional plates and a hollow transition zone, while euglenids only possess the proximal transitional plate. although the transition zone of most euglenids is also hollow, the transition zone in some euglenids, such as entosiphon applanatum and notosolenus mediocanellata, has been shown to be electron dense. however, the detailed structure of these transition zones still remains to be characterized in detail. the central area of the transition zone in c. aureus is also electron dense and contains a complex system of elements that have never been observed in any other euglenozoan so far. characterization of the flagellar transition zone in postgaardi might demonstrate several homologous elements that would help to further establish a close relationship between this lineage and c. aureus. nonetheless, diplonema ambulator, rhynchopus euleeides, r. coscinodiscivorus and c. aureus all have fibers that extend from each microtubular doublet to the flagellar membrane; these fibers have been called "transitional fibers". "transitional fibers" has also been used to describe fibers that extended from each microtubular triplet of a basal body to the flagellar membrane, which is potentially confusing. nonetheless, the "radial connectives" extending from the doublets in the transition zone of c. aureus are nearly identical, and likely homologous, to the'transitional fibers' extending from the doublets in diplonemids, such as d. ambulator. feeding apparatus each of the euglenozoan subgroups contains members with an elaborate feeding apparatus. most phagotrophic euglenids, for instance, have a distinctive feeding apparatus consisting of 4– central vanes and 2– supporting rods. some bacteriovorous euglenids, however, possess a much simpler feeding apparatus that is very similar to the mtr feeding pockets found in many kinetoplastids. the microtubules that support the rods in phagotrophic euglenids and the mtr pockets in bacteriovorous euglenozoans originate from the ventral root of the ventral basal body. similarly, the feeding pocket in c. aureus was also supported by microtubules that originated from the ventral root and is almost certainly homologous to the mtr pockets or rods found in other euglenozoans, including postgaardi. moreover, the compact "oblique striated fiber" that reinforces the feeding pocket in c. aureus was similar to the amorphous matrix found in some euglenid feeding rods and might represent a vestige of a more elaborate ancestral state. however, this inference will require improved understanding of the morphological diversity and phylogeny of other euglenozoans that are more closely related to c. aureus. a novel extrusomal pocket although tubular extrusomes are not widespread within the euglenozoa, several members from each main subgroup possess them, such as the euglenid entosiphon; the kinetoplastids rhynchobodo, hemistasia, and phylomitus; the diplonemid diplonema nigricans; and postgaardi mariagerensis. calkinsia aureus not only had tubular extrusomes like the lineages listed above, but they were clustered together much like the single battery of tubular extrusomes found in hemistasia. by contrast, postgaardi and rhynchobodo possess several smaller batteries of tubular extrusomes that are dispersed throughout the cytoplasm. the battery of tubular extrusomes in c. aureus was anchored to a novel extrusomal pocket that branched off of the vestibulum separately from the feeding apparatus and the flagellar apparatus. this battery of extrusomes was often discharged as a single unit from the extrusomal pocket and through the anterior opening. the functional significance of this process is unclear. the phagotrophic euglenid dinema sulcatum also contains a flagellar pocket and reportedly has two additional pockets: a "normal" feeding apparatus consisting of supportive rods and vanes and an "extra" pocket consisting of mtr-like microtubules. one previously proposed hypothesis for the presence of two feeding pockets in d. sulcatum involves the following inferences: the "extra" pocket is a remnant of the mtr feeding pocket present in the ancestral euglenozoan and the rod-and-vane based feeding apparatus represents a duplicated, and greatly embellished, mtr pocket that arose within a derived lineage of phagotrophic euglenids. this hypothesis is consistent with comparative morphological data that indicates other euglenid cytoskeletal components also evolved by duplication, such as the total number of pellicle strips around the cell periphery. nonetheless, the extrusomal pocket in c. aureus was supported by the lmt rather than microtubules from the ventral root, which support both mtr pockets and rod-and-vane based feeding apparatuses in euglenozoans. therefore, the extrusomal pocket in c. aureus appears to be novel and does not seem to be homologous to any type of feeding apparatus reported so far. euglenozoans with epibiotic bacteria postgaardi mariagerensis, euglena helicoideus, dylakosoma pelophilum, c. aureus and five unidentified euglenozoans from low oxygen environments have been reported to possess epibiotic bacteria on the cell surface. the epibiotic bacteria on d. pelophilum are spherical, and those on the other taxa are rod-shaped and densely packed on the cell surface. only one of the five unidentified euglenozoans, namely "morphotype c" from monterey bay, was studied with both sem and tem. the rod-shape epibiotic bacteria on these cells were not associated with a superficial distribution of mitochondrion-derived organelles beneath the host plasma membrane. nonetheless, morphotype c was clearly a euglenid, because the flagella contained paraxonemal rods, the feeding apparatus consisted of rods and vanes, and thin proteinaceous strips supported the cell surface. by contrast, the combination of ultrastructural features in c. aureus and p. mariagerensis make these lineages difficult to place within the euglenozoa. both lineages lack evidence of pellicle strips or kinetoplasts and possess paraxonemal rods, tubular extrusomes, mitochondrion-derived organelles beneath the plasma membrane, and condensed chromatin. detailed comparisons of the feeding apparatus in c. aureus, p. mariagerensis, and other anoxic euglenozoans should help better establish their phylogenetic relationships with each other; however, except for c. aureus, this information is currently lacking for nearly all of these lineages, including p. mariagerensis. molecular phylogenetic framework for euglenozoans in low-oxygen environments the morphology of c. aureus was completely concordant with the molecular phylogenetic data in so far as strongly placing c. aureus within the euglenozoa, but not with any of the three previously recognized subclades. figure shows the phylogenetic position of c. aureus within the euglenozoa, which consisted of five main clades. although petalomonas and notosolenus branched together as a separate clade, morphological evidence strongly supports their inclusion within the euglenida. therefore, the molecular phylogenetic data coupled with the morphological data allows us to recognize four clades of euglenozoans: the euglenida, the kinetoplastida, the diplonemida and a novel clade of anoxic euglenozoans, hereby named the symbiontida. the symbiontida includes several environmental sequences that were originally designated either as diplonemid sequences, as uncultured euglenozoan sequences or as "possible early branching eukaryotes". some of the environmental sequences within the symbiontida were already suspected to represent either a novel sister clade to the euglenozoa or novel subclade of euglenozoans. nonetheless, we have demonstrated that the symbiontida contains several more environmental sequences collected from different low-oxygen environments and also c. aureus, which provides an organismal anchor for this clade. we should also note that some environmental sequences from mid-atlantic hydrothermal vent environments in the "lost city", namely lc 5ep lc 5ep and lc 5ep grouped strongly with the diplonemid clade and not with the symbiotida. moreover, the lack of phylogenetic signal and perhaps also long-branch-attraction were the likely reasons for why the relatively fast-evolving sequences from notosolenus and petalomonas did not cluster strongly with the euglenid clade in our analyses of the dataset containing the shortest sequences. our analysis of the dataset including only the longest sequences, by contrast, clustered notosolemus and petalomonas with all other euglenids, albeit without strong statistical support. the symbiontida: a novel subclade of the euglenozoa before c. aureus had been studied at the ultrastructural and molecular phylogenetic levels, one author classified this lineage with p. mariagerensis within the taxon "postgaardea" on the basis of microaerophily. although our characterization of c. aureus has demonstrated epibiotic bacteria and mitochondrion-derived organelles like those described in p. mariagerensis, the presence of these characters in both lineages does not necessarily reflect homology. independently derived physical relationships between epibiotic bacteria and mitochondrion-derived organelles have been found in many different lineages of anoxic microeukaryotes, such as ciliates, oxymonads, parabasalids, heteroloboseans and euglenozoans. moreover, the presence of tubular extrusomes in both c. aureus and p. mariagerensis could be a symplesiomorphic state inherited from a very distant euglenozoan ancestor. nonetheless, our phylogenetic analyses demonstrate that c. aureus is a member of a newly recognized clade of anoxic euglenozoans consisting mainly of environmental sequences. the absence of molecular phylogenetic data and conclusive ultrastructural data from postgaardi precludes us from determining whether this lineage is also a member of the clade of microaerophiles. until these data are reported and the phylogenetic position of postgaardi is demonstrated more rigorously, we concur with a previous taxonomic treatment for postgaardi that recognizes this lineage as incertae sedis within the euglenozoa. as such, we conclude that it is premature to recognize the taxon postgaardea and view it as a synonym for p. mariagerensis. in light of the previous discussion, we propose the name "symbiontida" for the clade of microaerobic or anaerobic euglenozoans consisting of the most recent ancestor of c. aureus that also possessed rod-shaped epibiotic bacteria, reduced or absent mitochondrial cristae, tubular extrusomes and a nucleus with permanently condensed chromatin. this novel subclade of euglenozoans is recognized on the basis of ssu rdna-based phylogenetic data of lineages from several different low-oxygen environments. although the ultrastructural characteristics listed above are expected to be present in most, if not all, members of the symbiontida, this remains to be demonstrated with improved knowledge of euglenozoan diversity from both ultrastructural and molecular phylogenetic perspectives. phylogenetic diagnosis euglenozoa cavalier-smith 1981 symbiontida taxon nov. yubuki, edgcomb, bernhard & leander, 2009 apomorphy rod-shaped epibiotic bacteria above superficial layer of mitochondrion-derived organelles with reduced or absent cristae, homologous to the organization in calkinsia aureus, the type species. extended diagnosis of the type species calkinsia aureus lackey, emend., yubuki, edgcomb, bernhard & leander, 2009 paraxonemal rods present in flagella; kinetoplast dna and pellicle strips absent; long complex transitional zone between the basal bodies and the axonemes. rod-shaped epibiotic bacteria on perforated orange extracellular matrix. cell with a large nucleus on the anterior ventral side and a battery of tubular extrusomes linked to an extrusomal pocket located adjacent to the nucleus. feeding apparatus supported by both fibrous structures and microtubules that are derived from ventral root. small subunit ribosomal rna gene sequence distinguishes calkinsia aureus from all other symbiontid species.  molecular phylogenies inferred from ssu rdna demonstrate that c. aureus is closely related to several marine environmental sequences collected from low-oxygen environments, forming a novel subgroup within the euglenozoa, referred to here as the "symbiontida". improved understanding of these flagellates is necessary for further demonstrating the cellular identity of the symbiontida and for reconstructing the evolutionary radiation of the euglenozoan lineage. in this study, we characterized the detailed ultrastructure of c. aureus and demonstrated all of the euglenozoan synapomorphies and several cellular innovations associated with symbiotic interactions with epibiotic bacteria. we also demonstrated novel ultrastructural systems found in this species, such as the extrusomal pocket. environmental sequencing surveys from different low-oxygen environments around the world suggest that many symbiontid lineages have yet to be discovered and characterized. continued exploration into the overall diversity of this group should contribute significantly to our understanding of eukaryotic evolution, especially in low-oxygen environments. abbreviations af: anterior flagellum; b: epibiotic bacteria; ba: bacteriotroph; cgs: congregated globule structure; db: dorsal basal body; df: dorsal flagellum; dl: dorsal lamina; dr: dorsal root; e: extrusome; ep: extrusomal pocket; eu: eukaryotroph; ex: extracellular matrix; g: golgi body; gl: glycocalyx; if: intermediate fiber; il: ir-associated lamina; ir: intermediate root; lf: left fiber; lm: light microscope; lmt: linked microtubules; mtd: mitochondrion-derived organelle; n: nucleus; nu: nucleolus; os: osmotroph; osf: oblique striated fibrous structure; pf: posterior flagellum; ph: phototroph; rf: right fiber; s: somatonema; sbb: santa barbara basin; scb: sodium cacodylate buffer; sem: scanning electron microscope; vb: ventral basal body; t: tomentum; tem: transmission electron microscope; vf: ventral flagellum; vr: ventral root. authors' contributions ny carried out all of the lm, sem, tem and molecular phylogenetic work, wrote the first draft of the paper and participated in the collection of sediment samples from the sbb. vpe and jmb, the chief scientist, coordinated and funded the research cruise to the sbb. bsl funded and supervised the collection and interpretation of the ultrastructural and molecular phylogenetic data and contributed to writing the paper. all authors have read, edited, and approved the final manuscript. supplementary material additional file 1 maximum likelihood analysis of taxa focusing on the position of calkinsia aureus within the euglenozoa clade. two jakobids, andalucia incarcerata and a. godoyi, are used as outgroups in this analysis. the short environmental sequences are excluded from the dataset used in figure and fast-evolve euglenids sequences, ploeotia, menoidium and astasia, are included. ml bootstrap values greater than 50% are shown. thick branches indicate bayesian posterior probabilities over. ba, bacteriotroph; eu, eukaryotroph; os, osmotroph; ph, phototroph. genbank accession numbers of the sequences analyzed are shown in parentheses. click here for file additional file 2 maximum likelihood analysis of taxa focusing on the position of calkinsia aureus within the euglenozoa clade. two jakobids, andalucia incarcerata and a. godoyi, are used as outgroups in this analysis. the short environmental sequences are removed from the dataset used in figure and fast-evolve euglenids sequences, dinema, ploeotia, menoidium and astasia, are excluded. ml bootstrap values greater than 50% are shown. thick branches indicate bayesian posterior probabilities over. ba, bacteriotroph; eu, eukaryotroph; ph, phototroph. genbank accession numbers of the sequences analyzed are shown in parentheses. click here for file acknowledgements this work was supported by grants to bsl from the tula foundation, the national science and engineering research council of canada and the canadian institute for advanced research, program in integrated microbial biodiversity. funding for the collection of sediments and participation of vpe and jmb in this research was provided by the us national science foundation grant mcb- we also acknowledge the constructive feedback from four anonymous reviewers.
however, c. aureus also possessed several novel ultrastructural systems, such as modified mitochondria, an "extrusomal pocket", a highly organized extracellular matrix beneath epibiotic bacteria and a complex flagellar transition zone. improved understanding of these lineages is expected to shed considerable light onto the ultrastructure of prokaryote-eukaryote symbioses and the associated cellular innovations found within the euglenozoa and beyond. the group consists of three main subclades – euglenids, kinetoplastids and diplonemids – that have been confirmed with both molecular phylogenetic analyses and a combination of shared ultrastructural characteristics. molecular phylogenies inferred from ssu rdna sequences demonstrated that c. aureus grouped strongly within the euglenozoa and with several environmental sequences taken from low-oxygen sediments in various locations around the world. several poorly understood lineages of putative euglenozoans live in anoxic environments, such as calkinsia aureus, and have yet to be characterized at the molecular and ultrastructural levels. serial tem sections through individually prepared cells demonstrated that c. aureus shares derived ultrastructural features with other members of the euglenozoa. molecular phylogenetic analyses of c. aureus demonstrate that this lineage is a member of a novel euglenozoan subclade consisting of uncharacterized cells living in low-oxygen environments. our ultrastructural description of c. aureus establishes the cellular identity of a fourth group of euglenozoans, referred to as the "symbiontida". calkinsia aureus possesses all of the synapomorphies for the euglenozoa, but lacks traits that are specific to any of the three previously recognized euglenozoan subgroups. these biflagellates were distinctively orange in color and covered with a dense array of elongated epibiotic bacteria. we collected calkinsia aureus from core samples taken from the low-oxygen seafloor of the santa barbara basin, california.
 the euglenozoa is a large group of eukaryotic flagellates with diverse modes of nutrition. the group consists of three main subclades – euglenids, kinetoplastids and diplonemids – that have been confirmed with both molecular phylogenetic analyses and a combination of shared ultrastructural characteristics. several poorly understood lineages of putative euglenozoans live in anoxic environments, such as calkinsia aureus, and have yet to be characterized at the molecular and ultrastructural levels. improved understanding of these lineages is expected to shed considerable light onto the ultrastructure of prokaryote-eukaryote symbioses and the associated cellular innovations found within the euglenozoa and beyond.  we collected calkinsia aureus from core samples taken from the low-oxygen seafloor of the santa barbara basin, california. these biflagellates were distinctively orange in color and covered with a dense array of elongated epibiotic bacteria. serial tem sections through individually prepared cells demonstrated that c. aureus shares derived ultrastructural features with other members of the euglenozoa. however, c. aureus also possessed several novel ultrastructural systems, such as modified mitochondria, an "extrusomal pocket", a highly organized extracellular matrix beneath epibiotic bacteria and a complex flagellar transition zone. molecular phylogenies inferred from ssu rdna sequences demonstrated that c. aureus grouped strongly within the euglenozoa and with several environmental sequences taken from low-oxygen sediments in various locations around the world.  calkinsia aureus possesses all of the synapomorphies for the euglenozoa, but lacks traits that are specific to any of the three previously recognized euglenozoan subgroups. molecular phylogenetic analyses of c. aureus demonstrate that this lineage is a member of a novel euglenozoan subclade consisting of uncharacterized cells living in low-oxygen environments. our ultrastructural description of c. aureus establishes the cellular identity of a fourth group of euglenozoans, referred to as the "symbiontida". 