Fig. S1. Temperature dependence of nitrite production in HL72. Triplicate cultures of HL72 were incubated at the appropriate temperatures, nitrite concentrations were measured every 6–12 h and the maximal nitrite production rate calculated for each replicate. Values in the graph represent the mean of the maximal nitrite production rates calculated for each replicate. Errors bars indicate one standard deviation of the mean.

Fig. S2. Schematic representation of the genomic organization of amo genes in ‘Candidatus Nitrosocaldus yellowstonii’ compared with N. maritimus, C. symbiosum and environmental genome fragments from marine and soil Crenarchaeota.

Fig. S3. Structures of archaeal GDGT lipids present in HL72 and in sediments from Yellowstone National Park. Structures of archaeol (Ø; two molecules shown – one in black, another in grey), GDGT-II-IV, crenarchaeol (I and isomer VI) and compound VIII (trialkyl-type GDGT-II).

Fig. S4. Archaeal isoprenoid ether membrane lipids of HL72 grown under suboptimal conditions (72°C, pH 6.0). HPLC-MS base peak chromatogram of lipids extracted from a culture of HL72 grown at 72°C and pH 6.0. Roman numerals above peaks correspond to GDGT structures in Fig. S3. Note the higher relative abundance of trialkyl tetraether lipids (compounds VIII–XII) compared with the chromatogram in Fig. 5. The structure of VIII (trialkyl-type GDGT-II) was verified by MS/MS analysis. The molecular ion of VIII has an m/z of 1304 and fragmentation resulted in ions with m/z of 1024, 1006, 988, 950 and 932 representing a loss of one phytanyl group as phytene and subsequent losses of one or two glycerol moieties and water respectively.

Table S1. Physical and chemical properties of YNP sampling sites used for the detection of thermophilic AOA.

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