In our study, four of the six clones in OTU 18 were 100% identical to CSIRO-Qld19, a 16S rRNA gene sequence identified in the ovine rumen from Australia [30], and the single clone from OTU 38 was identical to ON-CAN.02, a 16S rRNA sequence identified in the bovine rumen from Canada [31]. Of the remaining alpaca sequences in this uncultured group, 16

of 24 clones had 98% or greater sequence identity to previously reported methanogen 16S rRNA genes isolated from rumen samples (data not shown). Figure 2 A neighbor-joining distance matrix tree of the archaea in the alpaca forestomach derived from 16S rRNA gene evolutionary distances produced by the Kimura two-parameter correction model [24]. Bootstrap supports are indicated as a percentage at the #check details randurls[1|1|,|CHEM1|]# base of each bifurcation. Bootstrap values less than 50% are

not shown. Evolutionary distance is represented by the horizontal component separating click here the species in the figure. The scale bar corresponds to 2 changes per 100 positions. Analysis of methanogen population structure in individual alpacas In the alpaca 4 library, 16S rRNA gene sequences were distributed between 21 of the 51 combined OTUs, with OTUs 1-5 representing 69.8% (125/179) of clones isolated from this individual (Table 1). We found that 57.5% (103/179) of sequences from alpaca 4 were grouped in OTUs showing 98% or greater sequence

identity to Methanobrevibacter millerae, while only 12.8% (23/179) were in OTUs that were categorized as unassigned Methanobrevibacter sequences (Table 3). Distinctively, alpaca 4 was the only individual for which we did not isolate any clones from the uncharacterized Decitabine ic50 archaeal group (OTUs 15, 18, 28, 31, 35, 38 and 48). In the alpaca 5 library, sequences were distributed between 27 OTUs, with OTUs 1, 3, 6, 7 and 12 representing the most clones obtained from this individual (66.3%, 132/199). Of note, 16S rRNA gene sequences from alpaca 5 showed the highest representation of unassigned Methanobrevibacter OTUs at 34.7% (69/199), as well as the highest representation in unassigned Methanobacterium OTUs at 13.1% (26/199) (Table 3). In addition, clones from this individual with species-level identity to Methanobrevibacter millerae were relatively under-represented at 32.7% (65/199) compared with alpacas 4, 6 and 9. In the alpaca 6 library, clones were found in 29 of 51 OTUs, the most within our sampled individuals, with 62.2% (125/201) divided among OTUs 1-5. Remarkably, 62.7% (126/201) of alpaca 6 sequences had species-level identity to Methanobrevibacter millerae, the highest representation from any individual, while only 7% (14/201) of its sequences had species-level identity to Methanobrevibacter ruminantium, the lowest representation in our study.