Detection and Phylogenetic Analysis of Novel Crenarchaeote and Euryarchaeote 16S Ribosomal RNA Gene Sequences from a Great Barrier Reef Sponge Nicole S. Webster, 1,3 Joy E.M. Watts, 2 and Russell T. Hill 1,2, * 1 Australian Institute of Marine Science, Townsville, Queensland, Australia, 4810 2 Center of Marine Biotechnology, University of Maryland Biotechnology Institute, MD 21202, USA 3 Department of Microbiology, James Cook University, Townsville, Queensland, Australia, 4814 Abstract: The presence of Archaea in the Great Barrier Reef marine sponge Rhopaloeides odorabile was inves- tigated by 16S ribosomal RNA community analysis of total DNA extracted from the sponge tissue. The 16S rRNA gene sequences corresponding to group I crenarchaeotes and group II euryarchaeotes were recovered from R. odorabile tissue. The location of archaeal cells within the sponge tissue was investigated using fluo- rescently labeled oligonucleotide probes. The presence of Archaea was confirmed within all regions of the sponge tissue from R. odorabile, with a significantly higher number of archaeal cells located in the pinacoderm than the mesohyl region. This is the first report of euryarchaeaotes associated with marine sponges. Key words: marine, sponge, Archaea, 16S rRNA, crenarchaeote, euryarchaeote. I NTRODUCTION Understanding of the community structure and global dis- tribution of Archaea has improved dramatically in recent years with the advent of molecular phylogenetic tools. The general phenotypic groups of Archaea were originally con- sidered to be the methanogens, the extreme halophiles, the sulfate-reducing Archaea, and the extreme thermophiles (DeLong, 1992). The 3 predominant catabolic themes em- ployed by Archaea are methanogenesis, heterotrophic oxi- dation of sugars and peptides (both aerobically and anaero- bically), and chemolithoautotrophic growth utilizing re- duced sulfur compounds or hydrogen. Archaea have predominantly been assigned to 2 kingdoms, the Crenar- chaeota and the Euryarchaeaota (Woese et al., 1990). More recently, a third kingdom, the Korarchaeota, was proposed to describe two 16S rRNA sequences retrieved from hot springs in Yellowstone National Park (Barns et al., 1996). Korarchaeota sequences branch deeply within the Crenar- chaeota, below the bifurcation between Crenarchaeota and Euryarchaeota. Cultured crenarchaeotes are extreme ther- mophiles and are generally obligate anaerobes with sulfur- dependent metabolism. Cultured euryarchaeotes include extreme halophiles, all known methanogens, and sulfur- metabolizing thermophiles. Archaeal 16S rRNA genes amplified from DNA from a range of marine samples have consistently clustered within one of two groups (DeLong, 1992). Marine group I is within the Crenarchaeota and marine group II is within the Eur- Received April 16, 2001; accepted June 27, 2001 *Corresponding author: telephone 410-234-8883; fax 410-234-8896; e-mail hillr@umbi.umd.edu Mar. Biotechnol. 3, 600–608, 2001 DOI: 10.1007/s10126-001-0065-7 © 2001 Springer-Verlag New York Inc.