Anaerobe 12 (2006) 134–139 Ecology/environmental microbiology Molecular identification of methanogenic archaea from sheep in Queensland, Australia reveal more uncultured novel archaea Andre´-Denis G. Wright Ã,1 , Andrew F. Toovey, Carolyn L. Pimm CSIRO Livestock Industries, Centre for Environment and Life Sciences, Private Bag 5, Wembley, W.A. 6913, Australia Received 25 October 2005; received in revised form 26 February 2006; accepted 28 February 2006 Available online 18 April 2006 Abstract Molecular diversity of rumen methanogens in sheep in Queensland, Australia was investigated using 16S rRNA gene libraries prepared from pooled rumen contents from nine merino sheep. A total of 78 clones were identified revealing 26 different sequences. Of these 26 sequences, eight sequences (15 clones) were 95–100% similar to cultivated methanogens belonging to the orders Methanobacteriales and Methanomicrobiales, and the remaining 18 phylotypes (63 clones) were 72–75% similar to Thermoplasma acidophilum and Thermoplasma volcanium. These unique sequences clustered within a distinct and strongly supported (100% bootstrap support) phylogenetic group, exclusively composed of sequences from uncharacterized archaea from very diverse anaerobic environments. Members of this unique group that were previously considered atypical for the rumen environment were the predominant clones. r 2006 Elsevier Ltd. All rights reserved. Keywords: Methanogens; Methanobrevibacter; Rumen ecology; Ruminants; Sheep 1. Introduction In Australia, ruminant livestock are the single largest source of methane emissions, accounting for 64% of agricultural methane emissions and at least 12% of Australia’s net emissions of carbon dioxide equivalents [1]. Methane is formed in the rumen when hydrogen released by other microbes during fermentation of forage is used by a distinct group of methane-producing archaea (i.e. methanogens) to reduce carbon dioxide. Methanogens play a significant part in the biological breakdown of organic matter in the digestive tracts of many vertebrates and invertebrates, and in other anaerobic environments includ- ing rice fields, and sewage treatment plants. Approximately 89% of the enteric methane emitted by ruminants is expired via the lungs [2] and exhaled at the nose and mouth. This loss of energy for the ruminant has been estimated to be between 2% and 12% of the animal’s gross energy intake [3]. A number of biological control options for decreasing ruminant methane emissions from grazing animals in Australia have been suggested [4], but the effectiveness of many of these options are dependent upon having an understanding of the numbers and, or, distribution of methanogen species among ruminants on different diets. Prior to the development of molecular-based methods, classical microbiological techniques were used to presump- tively identify methanogens from the digestive tracts of animals [5]. With the growing use of molecular techniques to investigate complex microbial systems, the application of these methods has proven to be very effective for the characterization of microbial communities, especially methanogen diversity in the rumen [6–14], and other diverse anaerobic environments [15–18]. In the present study, a combined 16S rRNA gene library was constructed from pooled rumen digesta samples from nine mature Merino sheep to determine the molecular diversity of methanogens from the ovine rumen in a tropical agricultural system in Queensland, Australia. ARTICLE IN PRESS www.elsevier.com/locate/anaerobe 1075-9964/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2006.02.002 Ã Corresponding author. Tel.: +61 7 3214 2200; fax: +61 7 3214 2900. E-mail address: andre-denis.wright@csiro.au (A.-D. Wright). 1 Present address: CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Rd., St. Lucia, Qld. 4067, Australia.