A phylogenomic and molecular markers based taxonomic framework for members of the order Entomoplasmatales: proposal for an emended order Mycoplasmatales containing the family Spiroplasmataceae and emended family Mycoplasmataceae comprised of six genera.

The "Spiroplasma cluster" is a taxonomically heterogeneous assemblage within the phylum Tenericutes encompassing different Entomoplasmatales species as well as the genus Mycoplasma, type genus of the order Mycoplasmatales. Within this cluster, the family Entomoplasmataceae contains two non-cohesive genera Entomoplasma and Mesoplasma with their members exhibiting extensive polyphyletic branching; additionally, the genus Mycoplasma is also embedded within this family. Genome sequences are now available for all 19 Entomoplasmataceae species with validly published names, as well as 6 of the 7 species from the genus Mycoplasma. With the aim of developing a reliable phylogenetic and taxonomic framework for the family Entomoplasmataceae, exhaustive phylogenetic and comparative genomic studies were carried out on these genome sequences. Phylogenetic trees were constructed based on concatenated sequences of 121 core proteins for this cluster, 67 conserved proteins shared with the phylum Firmicutes, 40 ribosomal proteins, three major subunits of RNA polymerase (RpoA, B and C) by different means and also for the 16S rRNA gene sequences. The interspecies relationships as well as different species groups observed in these trees were identical and robustly resolved. In all of these trees, members of the genera Mesoplasma and Entomoplasma formed three and two distinct clades, respectively, which were interspersed among the members of the other genus. The observed species groupings in the phylogenetic trees are independently strongly supported by our identification of 103 novel molecular markers or synapomorphies in the forms of conserved signature indels and conserved signature proteins, which are uniquely shared by the members of different observed species clades. To account for the different observed species clades, we are proposing a division of the genus Mesoplasma into an emended genus Mesoplasma and two new genera Tullyiplasma gen. nov. and Edwardiiplasma gen. nov. Likewise, to recognize the distinct species groupings of Entomoplasma, we are proposing its division into an emended genus Entomoplasma and a new genus Williamsoniiplasma gen. nov. Lastly, to rectify the long-existing taxonomic anomaly caused by the presence of genus Mycoplasma (order Mycoplasmatales) within the Entomoplasmatales, we are proposing an emendation of the family Mycoplasmataceae to include both Entomoplasmataceae plus Mycoplasma species and an emendation of the order Mycoplasmatales, which now comprises of the emended family Mycoplasmataceae and the family Spiroplasmataceae. The taxonomic reclassifications proposed here accurately reflect the species relationships within this group of Tenericutes and they should lead to a better understanding of their biological and pathogenic characteristics.

Acromyrmex Leaf-Cutting Ants Have Simple Gut Microbiota with Nitrogen-Fixing Potential.

Ants and termites have independently evolved obligate fungus-farming mutualisms, but their gardening procedures are fundamentally different, as the termites predigest their plant substrate whereas the ants deposit it directly on the fungus garden. Fungus-growing termites retained diverse gut microbiota, but bacterial gut communities in fungus-growing leaf-cutting ants have not been investigated, so it is unknown whether and how they are specialized on an exclusively fungal diet. Here we characterized the gut bacterial community of Panamanian Acromyrmex species, which are dominated by only four bacterial taxa: Wolbachia, Rhizobiales, and two Entomoplasmatales taxa. We show that the Entomoplasmatales can be both intracellular and extracellular across different gut tissues, Wolbachia is mainly but not exclusively intracellular, and the Rhizobiales species is strictly extracellular and confined to the gut lumen, where it forms biofilms along the hindgut cuticle supported by an adhesive matrix of polysaccharides. Tetracycline diets eliminated the Entomoplasmatales symbionts but hardly affected Wolbachia and only moderately reduced the Rhizobiales, suggesting that the latter are protected by the biofilm matrix. We show that the Rhizobiales symbiont produces bacterial NifH proteins that have been associated with the fixation of nitrogen, suggesting that these compartmentalized hindgut symbionts alleviate nutritional constraints emanating from an exclusive fungus garden diet reared on a substrate of leaves.