Multiple independent losses of sporulation and peptidoglycan in the and related orders of the class .

Many peptidoglycan-deficient bacteria such as the are known host-associated lineages, lacking the environmental resistance mechanisms and metabolic capabilities necessary for a free-living lifestyle. Several peptidoglycan-deficient and non-sporulating orders of interest are thought to be descended from Gram-positive sporulating through reductive evolution. Here we annotate 2650 genomes belonging to the class , according to the Genome Taxonomy Database, to predict the peptidoglycan and sporulation phenotypes of three novel orders, , and , known only through environmental sequence surveys. These lineages are interspersed between peptidoglycan-deficient non-sporulating orders including the and , and more typical Gram-positive orders such as the and . We use the extant genotypes to perform ancestral state reconstructions. The novel orders are predicted to have small genomes with minimal metabolic capabilities and to comprise a mix of peptidoglycan-deficient and/or non-sporulating species. In contrast to expectations based on cultured representatives, the order lacks many of the genes involved in peptidoglycan and endospore formation. The reconstructed evolutionary history of these traits suggests multiple independent whole-genome reductions and loss of phenotype via intermediate transition states that continue into the present. We suggest that the evolutionary history of the reduced-genome lineages within the class is one driven by multiple independent transitions to host-associated lifestyles, with the degree of reduction in environmental resistance and metabolic capabilities correlated with degree of host association.