**Phylogenomic Analyses and Molecular Signatures Elucidating the Evolutionary Relationships amongst the** *Chlorobia* **and** *Ignavibacteria* **Species: Robust Demarcation of Two Family-Level Clades within the Order** *Chlorobiales* **and Proposal for the Family** *Chloroherpetonaceae* **fam. nov**

**Sarah Bello <sup>1</sup> , Mohammad Howard-Azzeh <sup>2</sup> , Herb E. Schellhorn <sup>3</sup> and Radhey S. Gupta 1,\***


**Citation:** Bello, S.; Howard-Azzeh, M.; Schellhorn, H.E.; Gupta, R.S. Phylogenomic Analyses and Molecular Signatures Elucidating the Evolutionary Relationships amongst the *Chlorobia* and *Ignavibacteria* Species: Robust Demarcation of Two Family-Level Clades within the Order *Chlorobiales* and Proposal for the Family *Chloroherpetonaceae* fam. nov. *Microorganisms* **2022**, *10*, 1312. https://doi.org/10.3390/ microorganisms10071312

Academic Editors: Matthew Sattley and Robert Blankenship

Received: 7 April 2022 Accepted: 25 June 2022 Published: 29 June 2022

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**Abstract:** Evolutionary relationships amongst *Chlorobia* and *Ignavibacteria* species/strains were examined using phylogenomic and comparative analyses of genome sequences. In a phylogenomic tree based on 282 conserved proteins, the named *Chlorobia* species formed a monophyletic clade containing two distinct subclades. One clade, encompassing the genera *Chlorobaculum, Chlorobium, Pelodictyon,* and *Prosthecochloris,* corresponds to the family *Chlorobiaceae,* whereas another clade, harboring *Chloroherpeton thalassium, Candidatus* Thermochlorobacter aerophilum, *Candidatus* Thermochlorobacteriaceae bacterium GBChlB, and *Chlorobium* sp. 445, is now proposed as a new family (*Chloroherpetonaceae* fam. nov). In parallel, our comparative genomic analyses have identified 47 conserved signature indels (CSIs) in diverse proteins that are exclusively present in members of the class *Chlorobia* or its two families, providing reliable means for identification. Two known *Ignavibacteria* species in our phylogenomic tree are found to group within a larger clade containing several *Candidatus* species and uncultured *Chlorobi* strains. A CSI in the SecY protein is uniquely shared by the species/strains from this "larger *Ignavibacteria* clade". Two additional CSIs, which are commonly shared by *Chlorobia* species and the "larger *Ignavibacteria* clade", support a specific relationship between these two groups. The newly identified molecular markers provide novel tools for genetic and biochemical studies and identification of these organisms.

**Keywords:** phylogenomic and comparative genomic analyses; conserved signature indels (CSIs); molecular signatures; class *Chlorobia* and the families *Chlorobiaceae* and *Chloroherpetonaceae*; *Ignavibacteria*; uncultured species/strains related to *Chlorobia*/*Ignavibacteria*
