Fig. S1. Sampling sites within the abandoned Cae Coch mine: (A) mine roof (L1); (B) bacterial stalactite (arrowed, length ∼1.5 m; L2); and (C) the main stream flowing through the mine with acid streamer growths (stream width ∼0.5 m; L3); isolated pool (pH meter scale is 18 cm in height; L4).

Fig. S2. FISH analysis of: (A) and (C) an acid streamer (site L3M) and (B) a microbial stalactite (site L2) from Cae Coch. Micrographs (A1), (B1) and (C1) are strained with DAPI, and (A2), (B2) and (C2) with the fluorescein-labelled EUB338 probe. Sample (A3) is stained with the ‘Fv. myxofaciens’-specific probe BSC0459, sample (B3) with the Gallionella-like clone probe GALTS0084, and sample (C3) with the iron-oxidizing Acidithiobacillus probe TF539.

Fig. S3. FISH analysis of acid streamer and water samples from Cae Coch. (A) A streamer sample from site L3B stained with the fluorescein-labelled EUB338 probe (A1) and with the Cy3-labelled Sphingomonas-specific SPH120 probe (A2); (B) a streamer sample from site L3B stained with two probes targeting Firmicutes represented by clones L3B2C3 (B1) and L3B2C11 (B2); and (C) pool water sample L4B, targeted by the fluorescein-labelled EUB338 probe (C1) and the Acidimicrobium/Ferrimicrobium-specific probe ACM732 (C2).

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