Phylogenomic and molecular marker based studies to clarify the evolutionary relationships amongst Anoxybacillus species and demarcation of the family Anoxybacillaceae and some of its constituent genera

The family <i>Anoxybacillaceae</i> was recently proposed encompassing the genera <i>Anoxybacillus</i>, <i>Geobacillus</i>, <i>Parageobacillus</i>, <i>Saccharococcus</i> and <i>Thermolongibacillus</i>. Of these genera, <i>Anoxybacillus</i> contains >50% of the <i>Anoxybacillaceae</i> species. However, <i>Anoxybacillus</i> species form multiple unrelated clades in phylogenetic trees and their evolutionary relationships are unclear. To clarify the evolutionary relationships of <i>Anoxybacillus</i> and other <i>Anoxybacillaceae</i> species, detailed phylogenomic and comparative analyses were conducted on 38 <i>Anoxybacillaceae</i> species with available genomes. In a phylogenomic tree based on 1148 core proteins, all <i>Anoxybacillus</i>, <i>Geobacillus</i>, <i>Parageobacillus</i>, <i>Saccharococcus</i> and <i>Thermolongibacillus</i> species, excepting <i>Anoxybacillus sediminis</i>, formed a strongly supported clade representing the family <i>Anoxybacillaceae</i>. Five conserved signature indels (CSIs) reported here are also uniquely found in these species, providing robust means for the demarcation of family <i>Anoxybacillaceae</i> in molecular terms. In our phylogenomic tree and in the Genomic Taxonomy Database, <i>Anoxybacillus</i> species formed four distinct clades designated as <i>Anoxybacillus sensu stricto</i> (containing the type species <i>A. pushchinoensis</i>), <i>Anoxybacillus</i>_A, <i>Anoxybacillus</i>_B and <i>Anoxybacillus</i>_C. Our analyses have identified 17 novel CSIs which offer means to reliably distinguish species from these clades based upon multiple uniquely shared molecular characteristics. Additionally, we have identified three and seven CSIs specific for the genera <i>Geobacillus</i> and <i>Brevibacillus</i>, respectively. All seven <i>Brevibacillus</i>-specific CSIs are also shared by <i>Anoxybacillus sediminis</i>, which branches reliably with this genus. Based on the strong phylogenetic and molecular evidence presented here, we are proposing that the genus <i>Anoxybacillus</i> should be restricted to only the species from <i>Anoxybacillus sensu stricto</i> clade, whereas the species from <i>Anoxybacillus</i>_A, <i>Anoxybacillus</i>_B, and <i>Anoxybacillus</i>_C clades should be transferred into three novel genera <i>Anoxybacteroides</i> gen. nov., <i>Paranoxybacillus</i> gen. nov. and <i>Thermaerobacillus</i> gen. nov., respectively. Additionally, we are also proposing the transfer of <i>Anoxybacillus sediminis</i> to the genus <i>Brevibacillus</i>. The proposed changes, which reliably depict the evolutionary relationships among <i>Anoxybacillaceae</i> species, should be helpful in the studies of these organisms.