This is the README file for all supplementary data associated with: Wolfe JM, Fournier GP. Horizontal gene transfer constrains the timing of methanogen evolution. Nature Ecology and Evolution. Each file and its contents are described below. 1_LUT.csv Lookup table providing the clade and full species names for truncated names used in all subsequent files (required for analysis in downstream software). Alignment files: 2_smc_unmasked.fasta MUSCLE alignment of SMC sequences for Euryarchaeota, Cyanobacteria, and Aquificales. 3_smc_masked.fasta MUSCLE alignment of SMC sequences for Euryarchaeota, Cyanobacteria, and Aquificales. The alignment was subsequently masked with GUIDANCE, removing poorly aligned regions. 4_scpA_unmasked.fasta MUSCLE alignment of ScpA sequences for Euryarchaeota, Cyanobacteria, and Aquificales. 5_scpA_masked.fasta MUSCLE alignment of ScpA sequences for Euryarchaeota, Cyanobacteria, and Aquificales. The alignment was subsequently masked with GUIDANCE, removing poorly aligned regions. 6_scpB_unmasked.fasta MUSCLE alignment of ScpB sequences for Euryarchaeota and Cyanobacteria. 7_scpB_masked.fasta MUSCLE alignment of ScpB sequences for Euryarchaeota and Cyanobacteria. The alignment was subsequently masked with GUIDANCE, removing poorly aligned regions (note as in Table S2 in the paper, there are NO phylogenetically informative sites). 8_smc_307_cyanos.fasta MUSCLE alignment of SMC sequences for all 307 Cyanobacteria taxa available in GenBank, excluding sequences transferred from other bacterial clades. 9_ribosomal_cyanos.fasta MUSCLE alignment of 30 ribosomal proteins (detailed in Table S3 of the paper) for Cyanobacteria, to reconstruct species relationships. 10_ribosomal_euryarchaeota.fasta MUSCLE alignment of 30 ribosomal proteins (detailed in Table S3 of the paper) for Euryarchaeota, to reconstruct species relationships. 11_concatenated_smc_complex.fasta Concatenated MUSCLE alignment of SMC (file 3), ScpA (file 4), and ScpB (file 6) for Euryarchaeota, Aquificales, and Cyanobacteria. 12_concatenated_smc_complex_nohalos.fasta Concatenated MUSCLE alignment of SMC (file 3), ScpA (file 4), and ScpB (file 6) for Euryarchaeota, Aquificales, and Cyanobacteria. Species of Halobacteriales have been removed, as detailed in Table S1 of the paper. 13_meta_align_nohalos.fasta Concatenated MUSCLE meta-alignment with 3 partitions: 1) SMC complex (file 12) for Euryarchaeota and Cyanobacteria, 2) ribosomal genes for Cyanobacteria (file 9) and missing data for Euryarchaeota, 3) ribosomal genes for Euryarchaeota (file 10) and missing data for Cyanobacteria. Phylogenetic tree topologies: 14_smc_masked.tre RaxML gene tree of SMC alignment for Euryarchaeota, Cyanobacteria, and Aquificales (file 3). This topology is depicted in Fig. S2 of the paper. 15_scpA_unmasked.tre RaxML gene tree of ScpA alignment for Euryarchaeota, Cyanobacteria, and Aquificales (file 4). This topology is depicted in Fig. S3 of the paper. 16_scpB_unmasked.tre RaxML gene tree of ScpB alignment for Euryarchaeota and Cyanobacteria (file 6). This topology is depicted in Fig. S4 of the paper. 17_smc_307_cyanos.tre RaxML gene tree of SMC sequences for all 307 Cyanobacteria taxa available in GenBank, excluding sequences transferred from other bacterial clades (file 8). This topology is depicted in Fig. S1 of the paper. 18_ribosomal_euryarchaeota_species.tre RaxML species tree of ribosomal sequences for Euryarchaeota (file 10). 19_concatenated_smc_complex.tre RaxML gene tree of concatenated SMC, ScpA, ScpB for Euryarchaeota, Cyanobacteria, and Aquificales (file 11). 20_concatenated_smc_complex_nohalos_raxml.tre RaxML gene tree of concatenated SMC, ScpA, ScpB for Euryarchaeota, Cyanobacteria, and Aquificales (file 12). Species of Halobacteriales have been removed, as detailed in Table S1 of the paper. Bootstrap values from this tree are depicted in Fig. 1 of the paper. 21_concatenated_smc_complex_nohalos_pb.tre PhyloBayes gene tree of concatenated SMC, ScpA, ScpB for Euryarchaeota, Cyanobacteria, and Aquificales (file 12). Species of Halobacteriales have been removed, as detailed in Table S1 of the paper. This topology and its associated posterior probabilities are depicted in Fig. 1 of the paper. 22_meta_align_nohalos_raxml.tre RaxML tree topology reconstructed from meta-alignment (file 13) with 3 partitions: 1) SMC complex for Euryarchaeota and Cyanobacteria, 2) ribosomal genes for Cyanobacteria and missing data for Euryarchaeota, 3) ribosomal genes for Euryarchaeota and missing data for Cyanobacteria. Note this topology was input as a fixed tree in PhyloBayes divergence time estimates. 23_meta_align_nohalos_pb.tre PhyloBayes tree topology reconstructed from meta-alignment (file 13) with 3 partitions: 1) SMC complex for Euryarchaeota and Cyanobacteria, 2) ribosomal genes for Cyanobacteria and missing data for Euryarchaeota, 3) ribosomal genes for Euryarchaeota and missing data for Cyanobacteria. Divergence time estimates: 24_ribosomal_euryarchaeota_species.chronogram PhyloBayes chronogram with root prior only, with fixed topology from Euryarchaeota species tree (file 18). Select age CIs are depicted in the first column of Fig. 2A in the paper. 25_ribosomal_euryarchaeota_species.dates PhyloBayes dates file with root prior only, with fixed topology from Euryarchaeota species tree (file 18). 26_concatenated_smc_complex_nohalos.chronogram PhyloBayes chronogram with only an internal constraint on total-group Nostocales from an empirical fossil 1.2 Ga. The fixed topology is the concatenated SMC complex (file 21). Select age CIs are depicted in the middle column of Fig. 2A in the paper. 27_concatenated_smc_complex_nohalos.dates PhyloBayes dates file with only an internal constraint on total-group Nostocales from an empirical fossil 1.2 Ga. The fixed topology is the concatenated SMC complex (file 21). 28_root_only.chronogram PhyloBayes chronogram with root prior only on meta-alignment fixed topology (file 22). Select age CIs are depicted in first column of Fig. 2B in the paper. 29_root_only.dates PhyloBayes dates file with root prior only on meta-alignment fixed topology (file 22). 30_fossil_1.2.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from an empirical fossil 1.2 Ga. The fixed topology is the meta-alignment (file 22), and this is the same for all subsequent chronograms (files 32-52). This chronogram is depicted in Fig. 3 in the paper, as well as select age CIs in the last column of Fig. 2A (‘composite alignment’) and again in the second column of Fig. 2B. 31_fossil_1.2.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from an empirical fossil 1.2 Ga. The fixed topology is the meta-alignment (file 22), and this is the same for all subsequent dates files (files 33-53). 32_sim_fossil_1.3.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.3 Ga. Select age CIs are depicted in Fig. 2B in the paper. 33_sim_fossil_1.3.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.3 Ga. 34_sim_fossil_1.4.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.4 Ga. Select age CIs are depicted in Fig. 2B in the paper. 35_sim_fossil_1.4.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.4 Ga. 36_sim_fossil_1.5.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.5 Ga. Select age CIs are depicted in Fig. 2B in the paper. 37_sim_fossil_1.5.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.5 Ga. 38_sim_fossil_1.6.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.6 Ga. Select age CIs are depicted in Fig. 2B in the paper. 39_sim_fossil_1.6.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.6 Ga. 40_sim_fossil_1.7.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.7 Ga. Select age CIs are depicted in Fig. 2B in the paper. 41_sim_fossil_1.7.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.7 Ga. 42_sim_fossil_1.8.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.8 Ga. Select age CIs are depicted in Fig. 2B in the paper. 43_sim_fossil_1.8.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.8 Ga. 44_sim_fossil_1.9.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.9 Ga. Select age CIs are depicted in Fig. 2B in the paper. 45_sim_fossil_1.9.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from a simulated fossil 1.9 Ga. 46_fossil_2.0.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from an empirical fossil 2.0 Ga. Select age CIs are depicted in Fig. 2B in the paper. 47_fossil_2.0.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from an empirical fossil 2.0 Ga. 48_sim_fossil_2.1.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from a simulated fossil 2.1 Ga. Select age CIs are depicted in Fig. 2B in the paper. 49_sim_fossil_2.1.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from a simulated fossil 2.1 Ga. 50_sim_fossil_2.2.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from a simulated fossil 2.2 Ga. Select age CIs are depicted in Fig. 2B in the paper. 51_sim_fossil_2.2.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from a simulated fossil 2.2 Ga. 52_sim_fossil_2.3.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from a simulated fossil 2.3 Ga. Select age CIs are depicted in Fig. 2B in the paper. 53_sim_fossil_2.3.datesPhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from a simulated fossil 2.3 Ga. Supplemental results: 54_smc_masked_distances.xlsx Pairwise distances between tips in the SMC complex tree (file 21) extracted using T-REX. These form the y-axis of Fig. S5 in the paper. 55_meta_align_distances.xlsx Pairwise distances between tips in the meta-alignment tree (file 22) extracted using T-REX. These form the x-axis of Fig. S5 in the paper. 56_sequence_simulation_results.xlsx Age estimates resulting from sequence simulations for branch lengths and missing data blocks, as described in the supplemental appendix of the paper. Select age CIs are depicted in Figs. S6 and S7 in the paper. Effective prior files: 57_ribosomal_euryarchaeota_species.datedist PhyloBayes datedist file with root prior only, with fixed topology from Euryarchaeota species tree (file 18). 58_concatenated_smc_complex_nohalos.datedist PhyloBayes datedist file with only an internal constraint on total-group Nostocales from an empirical fossil 1.2 Ga. The fixed topology is the concatenated SMC complex (file 21). 59_fossil_1.2.datedist PhyloBayes datedist file with root prior and an internal constraint on total-group Nostocales from an empirical fossil 1.2 Ga. The fixed topology is the meta-alignment (file 22). 60_ribosomal_euryarchaeota_species_effective_prior.chronogram PhyloBayes chronogram with root prior only, under the effective prior (-prior flag), with fixed topology from Euryarchaeota species tree (file 18). 61_ribosomal_euryarchaeota_species_effective_prior.dates PhyloBayes dates file with root prior only, under the effective prior (-prior flag), with fixed topology from Euryarchaeota species tree (file 18). 62_ribosomal_euryarchaeota_species_effective_prior.datedist PhyloBayes datedist file with root prior only, under the effective prior (-prior flag), with fixed topology from Euryarchaeota species tree (file 18). 63_concatenated_smc_complex_nohalos_effective_prior.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from an empirical fossil 1.2 Ga, under the effective prior (-prior flag), with fixed topology from the concatenated SMC complex (file 21). 64_concatenated_smc_complex_nohalos_effective_prior.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from an empirical fossil 1.2 Ga, under the effective prior (-prior flag), with fixed topology from the concatenated SMC complex (file 21). 65_concatenated_smc_complex_nohalos_effective_prior.datedist PhyloBayes datedist file with root prior and an internal constraint on total-group Nostocales from an empirical fossil 1.2 Ga, under the effective prior (-prior flag), with fixed topology from the concatenated SMC complex (file 21). 66_fossil_1.2_effective_prior.chronogram PhyloBayes chronogram with root prior and an internal constraint on total-group Nostocales from an empirical fossil 1.2 Ga, under the effective prior (-prior flag), with fixed topology from the meta-alignment (file 22). 67_fossil_1.2_effective_prior.dates PhyloBayes dates file with root prior and an internal constraint on total-group Nostocales from an empirical fossil 1.2 Ga, under the effective prior (-prior flag), with fixed topology from the meta-alignment (file 22). 68_fossil_1.2_effective_prior.datedist PhyloBayes datedist file with root prior and an internal constraint on total-group Nostocales from an empirical fossil 1.2 Ga, under the effective prior (-prior flag), with fixed topology from the meta-alignment (file 22).