Molecular diversity of rumen methanogens from sheep in Western Australia

Abstract

The molecular diversity of rumen methanogens in sheep in Australia was investigated by using individual 16S rRNA gene libraries prepared from the rumen contents obtained from six merino sheep grazing pasture (326 clones), six sheep fed an oaten hay-based diet (275 clones), and five sheep fed a lucerne hay-based diet (132 clones). A total of 733 clones were examined, and the analysis revealed 65 phylotypes whose sequences (1,260 bp) were similar to those of cultivated methanogens belonging to the order Methanobrevibacter: Pasture-grazed sheep had more methanogen diversity than sheep fed either the oaten hay or lucerne hay diet. Methanobrevibacter strains SM9, M6, and NT7 accounted for over 90% of the total number of clones identified. M6 was more prevalent in grazing sheep, and SM9, despite being found in 16 of the 17 sheep, was more prevalent in sheep fed the lucerne-based diet. Five new species were identified. Two of these species exhibited very little sequence similarity to any cultivated methanogens and were found eight times in two of the six sheep that were grazing pasture. These unique sequences appear to represent a novel group of rumen archaea that are atypical for the rumen environment.

FIG. 1.

Phylogenetic relationships of the methanogens derived from 16S rRNA gene evolutionary distances produced by the Kimura two-parameter correction model (19) and constructed by using the neighbor-joining method (32). The consensus trees for 1,000 bootstrap resamplings of the data set were computed independently, and the bootstrap resampling values for 16S rRNA gene sequences (expressed as percentages) are indicated at the nodes. Bootstrap values less than 50% are not indicated. No significance should be placed on the lengths of the branches connecting the species.