Additional file 1 of Pre-Cambrian roots of novel Antarctic cryptoendolithic bacterial lineages

Additional file 1: Figure S1. Antarctic CBS form two distinct clades in the order Frankiales, with characteristic metabolic potential. a) Maximum-likelihood phylogenetic tree based on GTDB 120 core genes including representative genomes (violet) and Antarctic CBS (green). b) Principal Coordinate Analysis (Jaccard distance) of the protein cluster profiles (60% identity) b) and of the metabolic potential. d) The Fisher’s exact test (Bonferroni corrected p<0.01) highlights enriched functional categories. Genomes and KEGG orthologs are clustered according to the Hamming distance between the profiles. The top four KEGG categories significantly more present in the Antarctic CBS are highlighted in the upper bars. Figure S2. Antarctic Jiangellales CBS reveal a substantial genome reduction compared to known species, with characteristic differences in metabolic potential. a) Maximum-likelihood phylogenetic tree based on GTDB genes, including representative genomes from the GTDB database (violet) and Antarctic CBS (green). b) KEGG orthologs that are significantly less frequent in Antarctic Jiagellales compared to reference (uncorrected p<0.05, Fisher’s exact test). Only the first 25 pathways (ranked by the total number of significant orthologs) are shown. c) Number of predicted protein coding sequences in Antarctic (green) and reference (violet) Jiangellales d) The heatmap shows the presence (dark green) of KEGG orthologs belonging to the carotenoid biosynthesis pathway. The only gene involved in carotenoid biosynthesis detected in both CBS and GTDB reference genomes is the crtD. e) The phylogenetic tree inferred on the crtD gene highlights a segregation of Antarctic Jiangellales. Figure S3. Antarctic Thermomicrobiales ( class Chloroflexia ) CBS reveal characteristic metabolic potential. a) The Fisher’s exact test (uncorrected p<0.05) highlights a significant presence, in Antarctic genomes, of orthologs involved in transport, compared to the reference Thermomicrobiales genomes. Only the first 30 pathways (ranked by the total number of orthologs called significant) are shown. b) The prediction of protein coding sequences shows an increment of the number of genes in Antarctic Thermomicrobiales compared to reference genomes. Figure S4. Distribution of the number of CBS that are specific to a given number of samples, taxonomically classified at the Class level. We identified a set of 10 CBS (belonging to the classes Actinobacteria and Alphaproteobacteria) that are present in at least 75% (14/18) of the samples. Figure S5. Mash Screen was used to validate the presence of CBS in the Antarctic samples. a) Distribution of the number of CBS marked as present by the containment score estimated by Mash screen. 1009 out of 1094 (92.2%) CBS have been confirmed by Mash (containment score >0.95, green dashed vertical line). b) Distribution of the number of CBS marked as present by the estimated multiplicity. Figure S6. Scatter plot of the ANI estimated by mapping versus the containment scores estimated by Mash screen for each sample. Horizontal and vertical dashed lines represent the ideal species-level threshold of 0.95 for the containment score and the estimated ANI, respectively. Figure S7. a) Percentage of reads that could be mapped to the CBS representatives, grouped by Class. b) Per sample percentage of the reads that could be mapped to the CBS representatives, grouped by Class. Figure S8. The “ Candidatus Jiangella antarctica ” was found in each sample. a) Scatter plot of the ANI estimated by mapping versus the containment scores estimated by Mash screen (p < 1.47x10 -21 ). b) Scatter plot of the median depth of coverage estimated by mapping versus the median multeplicity estimated by Mash. The line of equality is represented in black. Supplementary Figure S9. Jaccard distance between the KEGG functional profiles for each Order.