The lower bound of the richness of the community was estimated with the nonparametric estimator CHAO1 using the software SPADE (version 3.1; Institute of Statistics, National Tsing Hua University http://chao.stat.nthu.edu.tw). The CHAO1 estimator was chosen according to the properties of the data set following the recommendations in the SPADE documentation. A Pareto-Lorenz evenness curve [65, 66] was used to illustrate and quantify the evenness of the Archaea community. The sequences were divided in OTUs based on a sequence similarity threshold of see more 98.7%
and ranked from high to low, based on their abundance. The cumulative proportion of OTU abundances (Y) was then plotted against the cumulative proportion of OTUs (X) resulting in a concave curve starting at (X, Y) = (0%, 0%) and ending in (X, Y) = (100%, 100%). The Fo index is the horizontal y-axis projection on the intercept with the vertical 20% x-axis line, i.e. the combined relative abundance of 20% of the OTUs. In a community with high evenness all or most OTUs are equally abundant which results in a Pareto-Lorenz curve close to a straight line of 45o. selleck inhibitor The Fo index for such a community is close to 20%. Specialized communities with one or a few dominating OTUs generate concave curves with high Fo indices. All sequences were compared with available sequences
in the GenBank nucleotide database using BLAST (Basic Local Alignment Search Tool)  Florfenicol August 22, 2011. The search tool of the SILVA rRNA database  was also used. However, matching sequences in GenBank always had higher similarities than the best matches from SILVA. TRF lengths were predicted for all clone library sequences. The sequences all started 50-100 bases away from the forward primer so the TRF lengths were predicted by alignment with a reference
sequence selleck products containing the primer site and assuming that there were no inserts or deletions between the primer and position 100. If the reference sequence had a restriction enzyme cut site preceding the first bases of the clone library sequence, the TRF for the clone library sequence could not be predicted. 25 sequences representing the 25 OTUs obtained by applying a sequence similarity threshold of 98.7% were subjected to phylogenetic analysis. The cloned sequences were aligned together with reference sequences representing known and proposed novel Archaea divisions using the alignment tool of the SILVA rRNA database . To make all sequences of equal length the resulting alignment was trimmed using BioEdit . Phylogenetic tree analysis was carried out using the PHYLIP package . Bootstrap analysis was carried out by generating 100 datasets using the program seqboot. The 100 datasets were analyzed by the maximum likelihood method using dnaml and 100 trees were created. The sequence of the bacteria Aquifex Pyrophilus was used as outgroup. A majority rule consensus tree was constructed from the 100 trees using consense.