Collection of organisms
Sediment samples were collected at low tide from the shoreline of Centennial Beach (Boundary Bay) in South-western British Columbia, Canada (49° 00' 4797''N, 123° 02' 1812''W), during the spring and summer of 2007 and 2008. The samples were taken at a depth of 1-3 cm below the sediment surface, from a conspicuous layer of black sand. The sediment samples were stored in flat containers at room temperature before individually isolated cells were prepared for light microscopy, electron microscopy and DNA extraction. Cells were extracted from the sand samples through a 48-μm mesh using the Uhlig melted seawater-ice method .
Attempts to culture the organism were made using two different media: ATCC 1728 (for growing Isonema) and CCAP 1259/1 (for growing Petalomonas cantuscygni). Both media were diluted in sterile seawater and kept under low oxygen conditions (oxygen content below 1%) using the ANAEROGEN™ COMPACT Kit system for anaerobic incubation; however, the cells did not reproduce and disappeared within 24 hours.
Light and electron microscopy
Differential interference contrast (DIC) light micrographs were taken using a Zeiss Axioplan 2 imaging microscope and a Leica DC500 digital chilled CCD camera.
Cells isolated from the British Columbia locality were fixed for scanning electron microscopy (SEM) using the 4% osmium tetroxide vapour protocol described previously . The cells were then transferred onto a 10-μm polycarbonate membrane filter, dehydrated with a graded ethanol series, and critical point dried with CO2 using a Tousimis Critical Point Dryer. The filter was then mounted on an aluminium stub, sputter coated with gold/palladium using a Cressington 208 HR High Resolution Sputter Coater, and observed with a Hitachi S-4700 field emission scanning electron microscope.
Cells isolated from the surrounding sediment were pre-fixed for transmission electron microscopy (TEM) using 4% (v/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (SCB) (pH 7.2) with the addition of 0.3 M sorbitol. The pre-fixed cells were washed in 0.2 M SCB (pH 7.2) three times and embedded in 2% of low melting temperature agarose and post-fixed in 1% (w/v) osmium tetroxide in 0.2 M SCB (pH 7.2) at room temperature for 1 hr, before being dehydrated through a graded series of ethanol and 100% acetone. The dehydrated cells were then infiltrated with acetone-Epon 812 resin mixtures and 100% Epon 812 resin. Ultra-thin serial sections were collected on copper Formvar-coated slot grids, stained with 2% (w/v) uranyl acetate and lead citrate, and observed using a Hitachi H7600 electron microscope.
DNA extraction, PCR amplification, alignment and phylogenetic analysis
Genomic DNA was extracted using the MasterPure Complete DNA and RNA purification Kit (Epicentre, WI, USA) from 30 cells that were individually isolated and washed three times in sterile seawater (i.e., "isolate 1"). This procedure was repeated three months later on a different sample of 30 individually isolated cells (i.e., "isolate 2"). Polymerase chain reactions (PCR) were performed using PuRe Taq Ready-To-Go PCR beads kit (GE Healthcare, Buckinghamshire, UK). Nearly the entire eukaryotic SSU rDNA gene was amplified from each isolate using the eukaryotic universal primers 5'- TGATCCTTCTGCAGGTTCACCTAC-3'  and 5'-GCGCTACCTGGTTGATCCTGCCAGT-3' . PCR amplifications consisted of an initial denaturing period (95°C for 3 min), 35 cycles of denaturing (93°C for 45 s), annealing (5 cycles at 45°C and 30 cycles at 55°C, for 45 s), extension (72°C for 2 min), and a final extension period (72°C for 5 min). The amplified DNA fragments were purified from agarose gels using UltraClean 15 DNA Purification Kit (MO Bio, CA, USA), and subsequently cloned into the TOPO TA Cloning Kit (Invitrogen, CA, USA). Two clones of the eukaryotic SSU rRNA gene, from each of the two isolates (i.e., four clones in total), were sequenced with the ABI Big-Dye reaction mix using the vector primers and internal primers oriented in both directions. The new sequences were screened with BLAST, identified by molecular phylogenetic analysis, and deposited into GenBank: HM004353, HM004354.
The SSU rDNA sequences from B. bacati were analyzed within the context of two alignments: (1) a 40-taxon alignment consisting of taxa representing all of the major groups of eukaryotes (988 unambiguously aligned sites) and (2) a 37-taxon alignment consisting of taxa representing all of the major lineages of euglenozoans (760 unambiguously aligned sites). Ambiguously aligned positions and gaps were excluded from both analyses. Phylogenetic relationships were inferred using maximum likelihood (ML) and Bayesian methods with the programs PhyML  and MrBayes , respectively. For ML, the nucleotide datasets were analysed using a general-time-reversible (GTR) model of base substitutions, plus a gamma correction with eight substitution rate categories and the proportion of invariable sites (GTR + I + G). ML bootstrap analysis of 500 replicates was performed with the same parameters described above. For Bayesian analyses, the program MrBayes was set to operate with a gamma correction with eight categories and proportion of invariable sites, and four Monte-Carlo-Markov chains (MCMC) (default temperature = 0.2). A total of 2,000,000 generations was calculated with trees sampled every 50 generations and with a prior burn-in of 100,000 generations (i.e., 2,000 sampled trees were discarded). A majority rule consensus tree was constructed from 18,000 post-burn-in trees with PAUP* 4.0. Posterior probabilities correspond to the frequency at which a given node is found in the post-burn-in trees.
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 (Cromwell Road, London, SW7 5BD, UK), American Museum of Natural History (Department of Library Services, Central Park West at 79th St., New York, NY, 10024, USA), Muséum national d'Histoire naturelle (Direction des bibliothèques et de la documentation, 38 rue Geoffroy Saint-Hilaire, 75005 Paris, France), Russian Academy of Sciences (Library for Natural Sciences of the RAS Znamenka str., 11, Moscow, Russia) and Academia Sinica (Life Science Library, 128 Sec. 2 Academia Rd, Nankang Taipei 115, Taiwan R.O.C.).
Formal Taxonomic Descriptions
Euglenozoa, Cavalier-Smith, 1981 
Symbiontida, Yubuki, Edgcomb, Bernhard & Leander, 2009 
Bihospitesn. gen. Breglia, Yubuki, Hoppenrath and Leander 2010
Uninucleate biflagellates; two heterodynamic flagella inserted subapically, with paraxial rods and no mastigonemes; flagella of approximately the cell length; elongated cells with a rounded posterior end; nucleus at anterior end of cell; cell covered with epibiotic bacteria of two different types: rod-shaped and spherical-shaped; cell surface with S-shaped folds; tubular extrusomes with cruciform core; presence of black bodies mainly at the anterior end of cell; rhythmic cell deformations and gliding motility.
Latin Bihospites, with two guests. The generic name reflects the presence of two different episymbiont morphotypes: rod-shaped, and spherical-shaped episymbionts.
Bihospites bacatin. sp. Breglia, Yubuki, Hoppenrath and Leander 2010
Cell elongated with rounded ends; cell size 40-120 μm in length and 15-30 μm in width; two heterodynamic flagella inserted subapically; anterior nucleus; cell covered with epibiotic bacteria of two different types: rod-shaped and spherical-shaped; cell surface with S-shaped folds; mitochondrion-derived organelles with reduced or absent cristae; feeding apparatus with conspicuous C-shaped rod and accessory rod that encircles the indented nucleus; the rod is formed by tightly packed, parallel-arranged lamellae; presence of black bodies, mainly at the anterior end of the cell; rhythmic cell deformations and gliding motility. Small subunit rRNA gene sequences [GenBank: HM004353, HM004354].
Both resin-embedded cells used for TEM and cells on gold sputter-coated SEM stubs have been deposited in the Beaty Biodiversity Research Centre (Marine Invertebrate Collection) at the University of British Columbia, Vancouver, Canada.
Figs 1A, 2A and 9A.
Tidal sand-flat at Centennial Beach, Vancouver, British Columbia, Canada (49°00' 4797''N, 123°02'1812''W).
Marine sand, black layer 2-3 cm deep.
Specific epithet, Latin bacati, ornamented with pearls. The etymology for the specific epithet reflects the presence of distinct longitudinal rows of spherical-shaped episymbionts, reminiscent of pearl necklaces.
Registration of new genus and species name in ZooBank
LSID for article: urn:lsid:zoobank.org:pub:40211D82-B95C-494A-B8D0-7E061E80DD18
LSID for the genus Bihospites: urn:lsid:zoobank.org:act:794D6C7B-BFB1-45C7-8DDA-32D44F3B0E50
LSID for the species B. bacati: urn:lsid:zoobank.org:act:E1549565-5434-4F85-B936-7D0C485596B8