Petalomonas sphagnophila is a poorly studied plastid-lacking euglenid flagellate living in Sphagnum-dominated peatlands. Here we present a broad-ranging microscopic, molecular and microspectrophotometric analysis of uncultured P. sphagnophila collected from four field locations in Nova Scotia, Canada. Consistent with its morphological characteristics, 18S ribosomal DNA (rDNA) phylogenies indicate that P. sphagnophila is specifically related to Petalomonas cantuscygni, the only other Petalomonas species sequenced to date. One of the peculiar characteristics of P. sphagnophila is the presence of several green-pigmented particles ∼5 μm in diameter in its cytoplasm, which a previously published study suggested to be cyanobacterial endosymbionts. New data presented here, however, suggest that the green intracellular body may not be a cyanobacterium but rather an uncharacterized prokaryote yet to be identified by molecular sequencing. 16S rDNA library sequencing and fluorescence in situ hybridizations show that P. sphagnophila also harbors several other endobionts, including bacteria that represent five novel genus-level groups (one firmicute and four different proteobacteria). 16S rDNA phylogenies suggest that three of these endobionts are related to obligate intracellular bacteria such as Rickettsiales and Coxiella, while the others are related to the Daphnia pathogen Spirobacillus cienkowskii or belong to the Thermoactinomycetaceae. TEM, 16S rDNA library sequencing and a battery of PCR experiments show that the presence of the five P. sphagnophila endobionts varies markedly among the four geographic collections and even among individuals collected from the same location but at different time points. Our study adds significantly to the growing evidence for complex and dynamic protist-bacterial associations in nature.
Bibliographical noteFunding Information:
This study was supported by postdoctoral fellowships to EK and JSP from the Centre for Comparative Genomics and Evolutionary Bioinformatics at Dalhousie University and the Tula foundation; an operating grant (ROP85016) awarded to JMA from the Canadian Institutes of Health Research (CIHR) regional Partnership Program, together with the Nova Scotia Health Research Foundation (NSHRF); NSERC Grant 298366-04 awarded to AGBS; Research Program of Hayama Center for Advanced Studies of Sokendai to SM; and Grant-in-Aid for Creative Scientific Research 17GS0134 from the Japan Society for the Promotion of Science to AM. We thank D Spencer and J Hopkins for help with sample collection, S Whitefield for assistance with light microscopy and P Colp for help with paraffin embedding and sectioning. B Leander is also thanked for assistance with interpretation of electron micrographs. JMA and AGBS are Fellows of the Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity, and JMA is a Canadian Institutes of Health Research New Investigator.