Single-cell genomics unveiled a cryptic cyanobacterial lineage with a worldwide distribution hidden by a dinoflagellate host Takuro Nakayamaa,1, Mami Nomurab,2, Yoshihito Takanoc, Goro Tanifujid, Kogiku Shibab, Kazuo Inabab, Yuji Inagakie, and Masakado Kawataa aGraduate School of Life Sciences, Tohoku University, Sendai 980-8578, Japan; bShimoda Marine Research Center, University of Tsukuba, Shimoda 415-0025, Japan; cFaculty of Science and Technology, Kochi University, Nankoku 783-8502, Japan; dDepartment of Zoology, National Museum of Nature and Science, Tsukuba 305-0005, Japan; and eCenter for Computational Sciences, University of Tsukuba, Tsukuba 305-8577, Japan Edited by David M. Karl, University of Hawaii at Manoa, Honolulu, HI, and approved May 24, 2019 (received for review February 13, 2019) Cyanobacteria are one of the most important contributors to revealed that this cyanobacterial lineage symbiotically interacts oceanic primary production and survive in a wide range of marine with a lineage of unicellular photosynthetic eukaryotes (14–17). habitats. Much effort has been made to understand their ecological This symbiosis is thought to trace back to at least the Late features, diversity, and evolution, based mainly on data from free- Cretaceous Period (18). Whole-genome sequencing of UCYN- living cyanobacterial species. In addition, symbiosis has emerged as A showed that the cyanobacterial lineage has greatly reduced an important lifestyle of oceanic microbes and increasing knowl- its metabolic capacities for photosynthesis and has been spe- edge of cyanobacteria in symbiotic relationships with unicellular cialized for nitrogen fixation, thus furthering current un- eukaryotes suggests their significance in understanding the global derstanding of marine cyanobacteria (16, 19). While detailed oceanic ecosystem. However, detailed characteristics of these genetic features of some symbiotic cyanobacteria have been cyanobacteria remain poorly described. To gain better insight into reported aside from UCYN-A (20–22), those of most cyano- marine cyanobacteria in symbiosis, we sequenced the genome of cyanobacteria collected from a cell of a pelagic dinoflagellate that bacterial symbionts remain unknown. These poorly understood ECOLOGY is known to host cyanobacterial symbionts within a specialized symbiotic species potentially have biodiversity, which is im- chamber. Phylogenetic analyses using the genome sequence revealed portant to an understanding of cyanobacteria as a whole. that the cyanobacterium represents an underdescribed lineage within Pelagic heterotrophic dinoflagellates of the genus Ornitho- an extensively studied, ecologically important group of marine cercus have long been known to host cyanobacteria as symbionts cyanobacteria. Metagenomic analyses demonstrated that this cyano- (Fig. 1). Ornithocercus species cells are surrounded by a cellulosic bacterial lineage is globally distributed and strictly coexists with its covering known as the thecal plate, and crown-shaped extensions host dinoflagellates, suggesting that the intimate symbiotic associa- tion allowed the cyanobacteria to escape from previous metagenomic Significance studies. Furthermore, a comparative analysis of the protein repertoire with related species indicated that the lineage has independently un- Cyanobacteria are an important component of marine micro- Prochloro- dergone reductive genome evolution to a similar extent as bial ecology, and thus their biodiversity has been extensively coccus , which has the most reduced genomes among free-living studied. Here, through whole-genome sequencing, we discov- cyanobacteria. Discovery of this cyanobacterial lineage, hidden by ered that a marine cyanobacterium in a symbiotic association its symbiotic lifestyle, provides crucial insights into the diversity, with a unicellular eukaryote (OmCyn) represents a previously ecology, and evolution of marine cyanobacteria and suggests the under-described lineage within an ecologically important cya- existence of other undiscovered cryptic cyanobacterial lineages. nobacterial group. Our metagenomic analyses showed that the cyanobacterium OmCyn thrives in global oceans, further sug- cyanobacteria | dinoflagellate | symbiosis | single-cell genomics | gesting the existence of other cryptic cyanobacterial lineages metagenomics that have been overlooked because of their symbiotic lifestyle. Via comparison with genomes of free-living relatives, the yanobacteria are one of the most successful groups of OmCyn genome was shown to have a reductive nature, which Coxygen-producing photoautotrophs occupying a broad range apparently resulted from intimate association with the host. of habitats on earth. Organisms in this group are highly ubiqui- Together, our results expand current understanding of the bi- tous in marine environments and play a vital role in oceanic ology of cyanobacteria and marine microbial ecology. biogeochemical cycles, with their metabolic abilities including photosynthesis and nitrogen fixation (1–3). To obtain a better Author contributions: T.N., M.N., Y.T., and Y.I. designed research; T.N., M.N., G.T., K.S., and K.I. performed research; T.N. analyzed data; and T.N., M.N., Y.T., G.T., K.S., K.I., Y.I., understanding of marine microbial ecology, the biodiversity and and M.K. wrote the paper. ecological features of marine cyanobacteria have been actively The authors declare no conflict of interest. explored (4–8). Previous studies based on environmental DNA and cultivated strains have revealed that marine cyanobacteria This article is a PNAS Direct Submission. display broad genetic diversity and have evolved by adapting to Published under the PNAS license. various ecological niches (9–11). Data deposition: The data reported in this paper are available from the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/), NCBI GenBank database (https://www.ncbi.nlm.nih. Other than free-living species, which have been extensively gov/genbank), and the European Molecular Biology Laboratory (https://www.embl.de/) studied, cyanobacterial species have symbiotic relationships with DNA database under BioProject accession number PRJDB7787. various organisms (12, 13). Recent developments in DNA-sequencing 1To whom correspondence may be addressed. Email: [email protected]. technologies have revealed the genetic characteristics of one group 2Present address: Graduate School of Human and Environmental Studies, Kyoto Univer- of symbiotic cyanobacteria. Unicellular cyanobacteria group A sity, Kyoto 606-8501, Japan. (UCYN-A; also known as Candidatus Atelocyanobacterium tha- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lassa) is a recently recognized, ecologically important cyano- 1073/pnas.1902538116/-/DCSupplemental. bacterial group in the marine environment. Recent studies have www.pnas.org/cgi/doi/10.1073/pnas.1902538116 PNAS Latest Articles | 1of6 Downloaded by guest on September 26, 2021 of the thecal plate form an extracellular chamber per cell (SI phylogenetic relationship between OmCyn and the marine Appendix, Fig. S1). A number of coccoid cyanobacteria reside in picocyanobacteria (SI Appendix, Fig. S3). The OmCyn sequence the specialized chamber (Fig. 1 and SI Appendix, Fig. S1); these made a monophyletic clade along with previously reported se- extracellular symbionts are also called phaeosomes (12, 23). quences. Most of those were a part of diverse 16S rDNA se- Despite the existence of phaeosomes being first recorded over quences directly amplified from dinoflagellate cells with 100 y ago (23), there is no laboratory culture or any report of cyanobacterial symbionts that are akin to O. magnificus (24). successful cultivation of the symbiont and detailed characteristics Biodiversity within the picocyanobacterial clade has been ex- of the cyanobacteria surviving in these chambers remain poorly tensively studied, as these cyanobacterial species numerically understood. Here, we sequenced the genome of the cyanobacteria dominate global oceans (7, 25). However, we detected no strong isolated from the dinoflagellate Ornithocercus magnificus (Fig. 1) phylogenetic affinity between the clade of cyanobacterial se- using single-cell genomics technology. Analyses based on the ge- quences from dinoflagellates, including OmCyn, and previously nome sequence revealed that the cyanobacterium represents an explicitly described marine picocyanobacterial lineages (7) based underdescribed lineage of a cyanobacterial clade, of which bio- on traditional phylogenetic markers for this group—namely, 16S diversity has been extensively studied and which has independently rDNA (SI Appendix, Fig. S3) and the internal transcribed spacer undergone reductive genome evolution. Analyses using meta- (ITS; SI Appendix, Fig. S4). The two trees showed different genomic data from the Tara Oceans Expedition further suggest phylogenetic positions of the clade comprising the cyanobacterial worldwide distribution of this cyanobacterial lineage and the po- sequences from dinoflagellates: The ITS tree suggested the clade tential existence of other undiscovered symbiotic cyanobacterial is basal to a well-defined Synechococcus clade, known as Syn- lineages. echococcus subcluster 5.1 (7), while the 16S rDNA tree placed the clade as sister to one of the clades within Synechococcus Results and Discussion subcluster 5.1, named clade V. Nevertheless, since both back- Genome Sequence and Phylogenetic Analyses of Cyanobacteria bones of the two trees did not receive high