Authors’ contributions AJM-R and JJF conceived and designed the experiments.

AJM-R conducted the experiments. AG-O and AH-C conducted the AFM work and processed the results from AFM measurements. AM conducted the CLSM work. RD-G carried out the statistical analysis. VSM and MN contributed with reagents, materials and valuable advice in the experimental design. AJM-R, AG-O, AH-C and JJF analysed the data. AJM-R and JJF wrote find more the paper. All authors read and approved the final manuscript.”
“Background Methanogen diversity has been widely investigated across a range of ruminants by using clone library sequence approaches and many unknown methanogen 16S rRNA sequences have been uncovered. Tajima et al. [1] investigated the diversity of bovine rumen fluid using two different

AZD8931 manufacturer archaea-specific primer sets, and for the first time reported the existence of a novel cluster of uncultured archaeal sequences which were distantly associated with Thermoplasma. However, the authors concluded that these novel sequences were likely from transient microbiota contaminating the animal feed, probably scavenging in an ecological niche in the rumen. Wright et al. [2] was the first to verify that these novel Thermoplasma-affiliated sequences were derived from the rumen when they investigated the diversity of rumen methanogens from sheep. The authors suggested a new order of methanogens for these novel sequences in the new cluster. The same authors [3] further found that over 80% of the total methanogen clones (63 of 78 clones) from the rumen of Merino sheep in Australia were 72–75% similar to Thermoplasmaacidophilum and Thermoplasmavolcanium. They [4] also found that about 50% of the total clones from methanogen 16S rRNA gene library PI-1840 of potato-fed DNA-PK inhibitor feedlot cattle were present in the new cluster, and 38% for corn-fed feedlot cattle. Huang et al. [5] found that Thermoplasmatales-affiliated sequences dominated in the yak and cattle methanogen clone libraries, accounting for 80.9% and 62.9% of the sequences in the two libraries, respectively. Our previous study [6] on the

diversity of methanogens in the rumen of Jinnan cattle showed that Thermoplasmatales-affiliated sequences were widely distributed in the rumen epithelium, rumen solid and fluid fractions. In addition, ruminant-derived sequences in this new cluster were also found in other studies [4, 7–12]. Based on the analysis of the global data set, Janssenand Kirs [13] placed the majority (92.3%) of rumen archaea detected in total rumen contents into three genus-level groups: Methanobrevibacter (61.6%), Methanomicrobium(14.9%), and a large group of uncultured rumen archaea affiliated with Thermoplasmatales (15.8%), and named the uncultured archaea group in the rumen, for the first time, as Rumen Cluster C (RCC). Using RCC specific DGGE, clone library analysis and quantitative real-time PCR, Jeyanathan et al.