69 [25]). MOTHUR was also used to generate a rarefaction curve, determine the Chao1 richness estimator, and calculate the Shannon and LIBSHUFF diversity indices. OTU coverage (C) was calculated using the equation C = 1-(n/N) × 100, where n is the number of OTUs represented by a single clone and N is the total number of clones analyzed in the library. Identification of representative OTU sequences was performed using the BLAST search engine http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi against

the NCBI nucleotide sequence database [26]. For phylogenetic reconstruction, 51 alpaca methanogen 16S rRNA sequences (one representative from each alpaca OTU) were combined with 45 methanogen 16S rRNA gene sequences representing major archaeal phylogenetic MK-8669 manufacturer groups. PHYLIP (Version 3.69 [25]) was used to construct a neighbor-joining tree [27], which was bootstrap resampled 1,000 times. Nucleotide sequence accession numbers The sequences from this study have been deposited in the GenBank database under the accession numbers JF301970-JF302647. For a detailed list of clones and accessions, see Additional file 1: Table S1. Results Phylogenetic analysis of methanogenic archaea in the alpaca forestomach We investigated

the diversity and phylogeny of methanogenic archaea in the forestomach of the alpaca by constructing individual methanogen 16S rRNA gene clone libraries from five animals. The number of non-chimeric clones isolated per individual library ranged from 179 to 201, for a combined total of 947 methanogen 16S rRNA gene sequences for analysis in our study. Based on a 98% find more sequence identity criterion, established from the level of identity that exists between 16S rRNA genes from validly characterized Methanobrevibacter species [6], our combined library sequences were grouped into 51 distinct OTUs (Table 1). Clones were unevenly distributed between OTUs, with 80.8% of sequences grouped within OTUs 1-10, compared with 19.2% for the remaining

41 OTUs. We used 2 different methods to assess the depth of coverage and sampling efficiency of our study at the OTU level. While the calculated rarefaction curve proved to be non-asymptotic, NADPH-cytochrome-c2 reductase it approached the saturation point (Figure 1), which we conservatively estimated to be 63 OTUs using the Chao1 richness indicator. Coverage (C) for individual and combined libraries was greater than 90% at the OTU level (Table 2). Together, these results support that the sampling efficiency of our study was very high. Table 1 OTU distribution of clones between individual alpaca animals OTU Nearest Valid Taxa % Seq. Identity Alpaca 4 Alpaca 5 Alpaca 6 Alpaca 8 Alpaca 9 Total Clones 1 Mbr. ruminantium 98.8 29 22 13 54 21 139 2 Mbr. millerae 98.1 27 15 49 12 7 110 3 Mbr. millerae 98.3 20 35 26 19 9 109 4 Mbr. millerae 99.0 33 1 16 4 55 109 5 Mbr. millerae 98.5 16 13 21 17 15 82 6 Mbr.