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

Abstract

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

Identity of epibiotic bacteria on symbiontid euglenozoans in O2-depleted marine sediments: evidence for symbiont and host co-evolution

“…Several morphological features of B. bacati are transitional between those found in C. aureus and those found in phagotrophic euglenids. Although these ultrastructural data suggest that the Symbiontida is nested within the Euglenida, current molecular phylogenetic data do not shed any light on this hypothesis (Breglia et al, 2010). C. aureus and B. bacati, share the feature of a coupling of rod-shaped epibionts with a superficial layer of hydrogenosome-like, mitochondrion-derived organelles having reduced or absent cristae.…”

“…Calkinsia aureus, B. bacati and Postgaardi mariagerensis have been isolated from oxygen-depleted marine environments; each one of them is covered with rod-shaped epibiotic bacteria (Simpson et al, 1997;Bernhard et al, 2000;Yubuki et al, 2009;Breglia et al, 2010). All three of these species have been characterized at the ultrastructural level, and C. aureus and B. bacati have also been characterized at the molecular phylogenetic level using small subunit rDNA sequences (Breglia et al, 2010;Simpson et al, 1997;Yubuki et al, 2009). The data from C. aureus and B. bacati showed a distinct subgroup of euglenozoans from oxygen-depleted environments (including seven environmental DNA sequences from Northern Europe and South America) referred to as the 'Symbiontida.'…”

“…The data from C. aureus and B. bacati showed a distinct subgroup of euglenozoans from oxygen-depleted environments (including seven environmental DNA sequences from Northern Europe and South America) referred to as the 'Symbiontida.' Symbiontid isolates and additional small subunit rDNA sequence representatives of the clade have now been recovered from seafloor sediments of Santa Barbara Basin, CA, USA coastal sediments of British Columbia, Canada, Northern Germany and anoxic and sulfidic waters in Venezuela, Denmark and Norway (Yubuki et al, 2009;Breglia et al, 2010).…”

“…bacati, which was recovered from oxygendepleted sandy sediments in a shallow tidal flat in South-western British Columbia, Canada, is 40-120 mm long and 15-30 mm wide and the cell surface is covered with two different morphotypes of epibionts: (1) spherical-shaped bacteria about 0.6 mm in diameter with an extrusive apparatus and (2) rod-shaped bacteria 3-5 mm long and arranged in bands along the longitudinal axis of the host (Breglia et al, 2010). Longitudinal bands of rod-shaped bacteria were separated by single or double rows of spherical-shaped bacteria.…”

“…It has also been hypothesized that the epibionts might be chemoautotrophic sulfur-or methane-oxidizers that form a mutualistic relationship with the host, whereby the host provides a substrate for the bacteria and the bacteria detoxify the immediate environment for the host (Bernhard, 2003;Bernhard et al, 2003Bernhard et al, , 2010. Under certain conditions, the epibiotic bacteria may serve as food for the host (Breglia et al, 2010). To better understand the evolutionary history of symbiotic relationships within the Symbiontida, we generated molecular data from the rod-shaped epibionts on both C. aureus and B. bacati.…”