A long-awaited taxogenomic investigation of the family Halomonadaceae

Abstract

The family Halomonadaceae is the largest family composed of halophilic bacteria, with more than 160 species with validly published names as of July 2023. Several classifications to circumscribe this family are available in major resources, such as those provided by the List of Prokaryotic names with Standing in Nomenclature (LPSN), NCBI Taxonomy, Genome Taxonomy Database (GTDB), and Bergey's Manual of Systematics of Archaea and Bacteria (BMSAB), with some degree of disagreement between them. Moreover, regardless of the classification adopted, the genus Halomonas is not phylogenetically consistent, likely because it has been used as a catch-all for newly described species within the family Halomonadaceae that could not be clearly accommodated in other Halomonadaceae genera. In the past decade, some taxonomic rearrangements have been conducted on the Halomonadaceae based on ribosomal and alternative single-copy housekeeping gene sequence analysis. High-throughput technologies have enabled access to the genome sequences of many type strains belonging to the family Halomonadaceae; however, genome-based studies specifically addressing its taxonomic status have not been performed to date. In this study, we accomplished the genome sequencing of 17 missing type strains of Halomonadaceae species that, together with other publicly available genome sequences, allowed us to re-evaluate the genetic relationship, phylogeny, and taxonomy of the species and genera within this family. The approach followed included the estimate of the Overall Genome Relatedness Indexes (OGRIs) such as the average amino acid identity (AAI), phylogenomic reconstructions using amino acid substitution matrices customized for the family Halomonadaceae, and the analysis of clade-specific signature genes. Based on our results, we conclude that the genus Halovibrio is obviously out of place within the family Halomonadaceae, and, on the other hand, we propose a division of the genus Halomonas into seven separate genera and the transfer of seven species from Halomonas to the genus Modicisalibacter, together with the emendation of the latter. Additionally, data from this study demonstrate the existence of various synonym species names in this family.

Keywords: genus delineation; halophiles; phylogenomics; signature genes; taxonomic reclassification.

Figure 1

Clade collapsed maximum-likelihood phylogenomic tree based on the concatenation of the translated sequence of 189 single-copy genes shared by at least 90% of the members of the family Halomonadaceae under study (“core90” set). The genus Pistricoccus is missing because no representative genome sequences from type strains of this genus were recovered. The number of species comprised within each clade is displayed. Ultrafast bootstrap values ≥70% are shown above the branches. Bar, 0.05 changes per position.

Figure 2

Box plot of the pairwise intra-clade and inter-clade AAI (A) and cAAI (B) values for the potential phylogroups/genera in the family Halomonadaceae, according to proposal I.

Figure 3

Gene family presence/absence patterns inferred from the pangenome of the family Halomonadaceae arranged according to proposal II. Each column represents a gene family pattern, where presence is indicated with a dot in the corresponding species. The absolute number of gene families that conform to each pattern is visualized in the marginal bar plot at the top. Separations between phylogroups/genera are indicated with horizontal black lines and the representative species of each phylogroup/genus is highlighted in bold. Genes that were present in all genomes of a clade and in none of the genomes outside of that clade, denoted as “signature genes,” are displayed in dark blue; other genes are shown in light blue. Patterns of presence in a single species or all species are not shown. The species Halovibrio salipaludis, Halovibrio variabilis, and Terasakiispira papahanaumokuakeensis were not considered members of the Halomonadaceae and were only used as an “outgroup”.

Figure 4

GTDB-derived maximum-likelihood phylogenomic tree based on the concatenation of 120 single-copy bacterial proteins (“bac120” set) showing the relationships among members of the family Halomonadaceae and related taxa. The type strain of the species Pistricoccus aurantiacus is missing because no genome sequences from this strain could be recovered. The number of species in each cluster is displayed inside wedges. Ultrafast bootstrap values ≥70% are shown above the branches. Bar, 0.05 changes per amino acid position.

Figure 5

ANI values (%) from pairwise whole-genome comparisons between the type strains of members of the family Halomonadaceae. The ANI percentages are expressed as a heatmap in purple color. Values supporting the proposed synonym species names are surrounded by a blue triangle.