Proc. Natl. Acad. Sci. USA Vol. 93, pp. 6241-6246, June 1996 Evolution A psychrophilic crenarchaeon inhabits a marine sponge: Cenarchaeum symbiosum gen. nov., sp. nov. (Archaea/Crenarchaeota/evolution/symbiosis/Porifera) CHRISTINA M. PRESTON*, KE YING Wu*, TADEUSZ F. MOLINSKIt, AND EDWARD F. DELONG*t *Department of Ecology, Evolution, and Marine Biology, University of California, Santa Barbara, Santa Barbara, CA 93106; and tDepartment of Chemistry, University of California, Davis, Davis, CA 95616 Communicated by Carl R. Woese, University of Illinois, Urbana, IL, February 2, 1996 ABSTRACT Archaea, one of the three major domains of growth on reduced sulfur compounds or hydrogen, represent extant life, was thought to comprise predominantly microor- three of the major catabolic themes (6). Limited capacity for ganisms that inhabit extreme environments, inhospitable to phototrophic energy generation, nitrogen fixation, and deni- most Eucarya and Bacteria. However, molecular phylogenetic trification has also been demonstrated in various archaeal surveys of native microbial assemblages are beginning to groups. Of the two major branches of Archaea, cultivated indicate that the evolutionary and physiological diversity of Crenarchaeota, comprised predominantly of anaerobic ther- Archaea is far greater than previously supposed. We report mophiles metabolizing elemental sulfur, have appeared the here the discovery and preliminary characterization of a most limited phenotypically (7). marine archaeon that inhabits the tissues ofa temperate water Recent molecular phylogenetic surveys of bacterial and ar- sponge. The association was specific, with a single crenar- chaeal diversity in cold oxygenated ocean waters (8-11) and chaeal phylotype inhabiting a single sponge host species. To terrestrial soils (12) have suggested the existence of additional our knowledge, this partnership represents the first described crenarchaeal phylotypes. Unfortunately, these unusual archaea symbiosis involving Crenarchaeota. The symbiotic archaeon have to date resisted laboratory cultivation, and so their pheno- grows well at temperatures of 10°C, over 60°C below the typic and physiological properties remain unknown. A low- growth temperature optimum of any cultivated species of growth temperature range for marine planktonic archaea has Crenarchaeota. Archaea have been generally characterized as been inferred from circumstantial evidence-e.g., their ecologi- microorganisms that inhabit relatively circumscribed niches, cal distribution and the relatively low percent G+C content of largely high-temperature anaerobic environments. In con- their ribosomal RNA genes (8-11). Nevertheless, it has not been trast, data from molecular phylogenetic surveys, including conclusively demonstrated that these microorganisms actually this report, suggest that some crenarchaeotes have diversified grow in cold habitats and do not instead emanate from alloch- considerably and are found in a wide variety of lifestyles and thonous sources (e.g., hydrothermal environments). habitats. We present here the identification and initial de- We recently surveyed microbial populations associated with scription of Cenarchaeum symbiosum gen. nov., sp. nov., a marine sponges by these new molecular methods (13). In the symbiotic archaeon closely related to other nonthermophilic course of that study, we detected significant amounts of archaeal crenarchaeotes that inhabit diverse marine and terrestrial nucleic acids associated with the tissues of a single marine sponge environments. species. We present here the identification and initial description of the sponge-associated archaeon and discuss the general im- Phylogenetic comparisons of universal, highly conserved mac- plications of our observations for archaeal biology. romolecular features of diverse biota have resulted in funda- mental conceptual changes in evolutionary biology. In partic- ular, the recent elucidation of evolutionary relationships METHODS among disparate life forms has revealed that prokaryotes Field Collection, Sponge Maintenance, and Identification. (defined as microorganisms lacking a membrane-bound nu- Sponges were collected at depths of 10-20 m by SCUBA diving cleus) consist of two evolutionarily distinct lineages (1-4). at two different sites located just offshore of Santa Barbara, Current data suggest that contemporary life on Earth has CA. The encrusting sponges were removed along with a small diverged into three major phylogenetic lineages (1-5), desig- amount of their rock substrate, placed in collection bags, and nated domains (5), two ofwhich are prokaryotic (Bacteria and transported to aquaria within 2 hr. Captive specimens were Archaea) and one of which is eukaryotic (Eucarya). Archaea, maintained in flowing seawater tanks. Criteria used in sponge the most recently recognized of the domains, contains culti- specimen identification included gross morphology, color, vated members that span a fairly limited range of phenotypes, oscule arrangement, encrusting shape and thickness, and the represented by extreme halophiles, sulfur-metabolizing ther- size and shape of siliceous spicules, which included both oxea mophiles, thermophilic sulfate-reducers, and methanogens and style types. No microscleres or ectosomal skeleton were (6). Although the number of new species of cultivated Archaea present, and the choanosomal skeleton was halichondroid-like has increased dramatically in recent years (7), the variety of (Mary Kay Harper and Welton Lee, personal communication). phenotypic motifs found within the Archaea has not. Virtually The sponge harboring the archaeal symbionts represents a all new archaeal isolates can still be categorized into one of the single species and was most similar toAxinella mexicana, based four commonly encountered phenotypes listed above. on the morphological criteria listed above and by comparison In contrast to those of Bacteria, the major physiological to type specimens (Welton Lee, personal communication). motifs of known and cultivated Archaea have appeared lim- Because of small differences in size and skeletal ited. Methanogenesis, aerobic or anaerobic heterotrophic ox- spicule idation of sugars and peptides, and chemolithoautotrophic Abbreviations: ssu, small subunit; DAPI, 4',6-diamidino-2-phenylindole. Data deposition: The sequence reported in this paper has been The publication costs of this article were defrayed in part by page charge deposited in the GenBank data base (accession no. U51469). payment. This article must therefore be hereby marked "advertisement" in 4To whom reprint requests should be addressed. e-mail: delong@ accordance with 18 U.S.C. §1734 solely to indicate this fact. marbtech.lscf.ucsb.edu. 6241 Downloaded by guest on September 26, 2021 6242 Evolution: Preston et al. Proc. Natl. Acad. Sci. USA 93 (1996) arrangement, however, this sponge may represent a new the purified double-stranded plasmid was directly sequenced. species and will therefore be referred to as "Axinella sp." Maximum likelihood analysis (19) was performed on a total of throughout this report. 1189 nucleotide positions, with the software FASTDNAML, Nucleic Acid Extraction and Hybridization. A vertical cross version 1.0 (20) using empirical base frequency, global branch section of sponge (0.5 g) was mechanically dissociated in swapping, and bootstrapping options. 0.22-gm filtered, autoclaved seawater using a tissue homoge- Whole Cell Hybridization. A vertical cross section of a nizer. Cell lysis was accomplished by incubating the dissociated captive sponge was fixed for 7 hr at 4°C in 3.7% formaldehyde cells in 1 mg of lysozyme per ml for 30 min at 37°C followed diluted in 0.22 ,um-filtered, autoclaved seawater. The section by an incubation for 30 min at 55°C with 0.5 mg of proteinase was subsequently rinsed in sterile seawater and macerated with K per ml and 1% SDS. The tubes were finally placed in a a tissue grinder. Spicules and large aggregates of sponge tissue boiling water bath for 60 sec to complete lysis. The protein were removed by low-speed centrifugation before spotting fraction was removed with two extractions with phenol:chlo- onto gelatin-subbed slides. To reduce background, the slides roform:isoamyl alcohol (50:49:1), pH 8.0, followed by a chlo- were treated with acetic anhydride (21). Slides were dehy- roform:isoamyl alcohol (24:1) extraction. Nucleic acids were drated in an ethanol series (50%, 75%, and 95%, 2 min each) ethanol-precipitated and resuspended in TE buffer (10 mM and air-dried (22). Hybridization conditions were as described Tris-HCl/1 mM Na2-EDTA, pH 8.0). Approximately 5 ,tg of (23, 24) but without the addition of bovine serum albumin. A DNA was purified by CsCl equilibrium density gradient ultra- mixture of four marine crenarchaeal-specific fluor-labeled centrifugation on a Beckman Optima tabletop ultracentrifuge oligonucleotide probes (5 ng/gl each) was added, and hybrid- using a TLA100 rotor (9). izations were incubated at 40°C overnight in a moist chamber For RNA extractions, vertical cross sections of tissue (150 mg) to prevent evaporation. After hybridizaton, slides were washed were removed, and total rRNA was extracted in hot phe- in lx SET (150 mM NaCl/20 mM Tris-HCl, pH 7.8/1 mM nol:CHCl3, pH 5.1 as described (14), with the following modifi- Na2-EDTA) at 45°C for 10 min, stained with 10 ,tg of cations. The sponge section was immersed in 0.7 ml of 50 mM 4',6-diamidino-2-phenylindole (DAPI) per ml for 5 min at sodium acetate buffer (pH 5.1), 0.7 ml of phenol