2.14. Construction of Phylogenetic Trees

Maximum likelihood 16S rRNA gene sequence-based phylogeny was computed for the novel strains, all described planctomycetal species of the order Pirellulales [28,29,30,31,40,42,61,62,63,64,65,66,67,68,69,70,71,72,73] and an outgroup of three strains from the order Planctomycetales (acc. no. {"type":"entrez-nucleotide","attrs":{"text":"MK554524","term_id":"1579174589","term_text":"MK554524"}}MK554524, {"type":"entrez-nucleotide","attrs":{"text":"MK554557","term_id":"1579174624","term_text":"MK554557"}}MK554557, and {"type":"entrez-nucleotide","attrs":{"text":"X62911","term_id":"45551","term_text":"X62911"}}X62911). The 16S rRNA gene sequences were aligned with SINA [58]. The phylogenetic analysis was then done by employing a maximum likelihood approach with 1000 bootstraps, the nucleotide substitution model GTR, gamma distribution and estimation of proportion of invariable sites (GTRGAMMAI option) [74].

For the multilocus sequence analysis (MLSA), the unique single-copy core genome of all analyzed genomes was determined with proteinortho5 [75] with the ‘selfblast’ option enabled, a coverage of 50%, and an e-value of 1e-05. The protein sequences of the resulting orthologous groups were aligned using MUSCLE v.3.8.31 [76]. After clipping, partially aligned C- and N-terminal regions and poorly aligned internal regions were filtered using Gblocks [77]. The final alignment of 570 ubiquitous genes with a combined length of 292,572 conserved amino acid residues was concatenated and clustered using FastTree [78]. The outgroup consisted of three genomes from strains of the order Planctomycetales (acc. no. {"type":"entrez-nucleotide","attrs":{"text":"CP036342","term_id":"1712542717","term_text":"CP036342"}}CP036342, {"type":"entrez-nucleotide","attrs":{"text":"CP036347","term_id":"1712562588","term_text":"CP036347"}}CP036347, and {"type":"entrez-nucleotide","attrs":{"text":"CP001744","term_id":"296012614","term_text":"CP001744"}}CP001744). Visualizations were performed with iToL v.4 [79].

Members of the novel genus Roseiconus [73] clustered within the earlier described genus Stieleria [40,62]. The results for both genera are therefore summarized under the latter term for all analyses.