Fig. 1: Visualization and phylogenetic affiliation of ‘Ca. A. ciliaticola’ and its ciliate host.

a, Representative scanning electron microscopy image of a ciliate from the anoxic hypolimnion of Lake Zug (February 2020, depth of 186 m). Scale bar, 5 μm. b–d, Differential interference contrast image (b) and confocal laser scanning microscopy image (c) of a ciliate after hybridization with a ‘Ca. Azoamicus’-specific oligonucleotide probe (eub62A3_813) (yellow) and counterstaining with DAPI (blue). d, Overlay of fluorescence and differential interference contrast images. The macronucleus (MAC) with attached micronucleus (arrowhead) and putative food vacuoles of the ciliate are outlined. Scale bars, 5 μm. e, 16S rRNA gene sequence-based maximum likelihood phylogenetic tree of ‘Ca. A. ciliaticola’ (bold denotes sequence derived from circular metagenome-assembled genome) and members of related gammaproteobacterial orders and environmental clades. Subgroups A and B of the ‘Ca. Azoamicus’ group are indicated. The sequence similarities of respective groups to the 16S rRNA gene sequence of ‘Ca. A. ciliaticola’ are shown in parentheses. The full tree is shown in Extended Data Fig. 4. Taxonomic groups were assigned on the basis of SILVA taxonomy⁴⁵. Metag., metagenome. f, Ciliate 18S rRNA gene sequence-based maximum likelihood phylogenetic tree of the class Plagiopylea based on EukRef-Ciliophora reference alignment³⁰. Sequences of ciliates from Lake Zug (in bold) were amplified from individual ciliates (S1–S4) or combined ciliates (C5 and C10). Stars denote positive identification of a ‘Ca. A. ciliaticola’ 16S rRNA sequence amplified from the same ciliate: black, ‘Ca. A. ciliaticola’ 16S rRNA gene sequence verified by Sanger sequencing; grey, positive band on gel. For both trees, bootstrap values are shown as circles at the respective nodes, and indicate bootstrap support of >70% (grey) or >90% (black) out of 100 resamplings. Scale bars, 0.05 nucleotide substitutions per site.