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Fig. S1. The LCMS ITS region encodes a tRNA and shows sequence similarity to the ITS regions of several methanogens. The alignment includes sequences from: Methanosaeta thermophila (NC_008553), Methanosarcina barkeri (NC_007349), Ms. acetivorans (NC_003552), Ms. mazei (NC_003901), Methanococcoides burtonii (NC_007955), Methanobacterium thermoautotrophicus (NC_000916), Methanosphaera stadtmanae (NC_007681), and Lost City Methanosarcinales (GQ273207).

Fig. S2. Rank-abundance plot showing the number of clones sharing the 10 most frequently occurring 16S rRNA and ITS sequences in samples LC0424 and LC1408, both of which were collected from the Poseidon chimney (Marker 3). Only one 16S rRNA sequence occurs more than twice, but five ITS sequences occur many times in this sample. As shown in Fig. 3B, other samples contain different abundant ITS sequences.

Table S1. Previously published characteristics of the three carbonate chimney samples from which V6 tags and V6-ITS clone libraries were sequenced. Fluid temperatures and concentrations of H2 and CH4 are maximum values reported by Proskurowski and colleagues (2006; 2008). Cell densities and proportions of phylogenetic groups are from Schrenk and colleagues (2004) and M. Schrenk (doctoral dissertation, 2005). Organic carbon concentrations and isotopic measurements are from Bradley and colleagues (2009). Fluid temperature and chemistry are identical for samples LC1404 and LC1443 because these carbonate samples were collected from the same chimney.

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