Keller-Costa, Tina

Abstract Members of the bacterial family Endozoicomonadaceae are ubiquitous marine symbionts associated with diverse hosts, including corals, sponges, molluscs, and fishes. Despite their ecological pervasiveness, inhabiting hosts in deep-sea vents and sunlit coral reefs, their taxonomy remains inconsistent, largely due to reliance on 16S rRNA gene phylogenies that fail to fully resolve evolutionary relationships. Here, we integrate phylogenomics, comparative genomics, and overall genome relatedness indices across 63 high-quality genomes to re-define genus boundaries within the Endozoicomonadaceae family. Among the metrics, the core-proteome average amino acid identity proved the most robust for genus-level resolution, with a proposed identity cutoff of 76%, aligning with phylogenomic structure and maintaining taxonomic robustness across the family. Phylogenomic reconstructions further revealed that the taxon Endozoicomonas is paraphyletic, forming two well-supported but distinct clades. This prompts a taxonomic revision, dividing Endozoicomonas into Endozoicomonas sensu stricto and Neoendozoicomonas gen. nov., with Neoendozoicomonas montiporae designated as the type species. Furthermore, the sister clade to Endozoicomonas sensu stricto—comprising the genera Endonucleibacter and Sororendozoicomonas—is characterized by genomic streamlining, including reduced genome size, lower GC content, reduced number of orthogroups, and potential functional divergences consistent with host specialization. Functional annotations highlighted secretion and conjugation systems as key differentiators between genera, emphasizing potential distinct host-interaction strategies. This genome-based framework refines the taxonomy of the Endozoicomonadaceae, provides additional criteria for genus delimitation, and strengthens the evolutionary and ecological interpretation of these widespread marine bacterial symbionts.

This study describes a Gram-stain-negative, rod-shaped, facultatively anaerobic bacterial species isolated from the octocoral Litophyton sp. inhabiting the live coral aquarium at Oceanário de Lisboa in Portugal. Four strains, NE35, NE40T, NE41 and NE43, were classified into the genus Endozoicomonas by means of 16S rRNA gene and whole-genome sequence homologies. We then performed phylogenetic, phylogenomic and biochemical analyses to examine their novel species status within the Endozoicomonas genus, based on comparisons with the designated novel type strain NE40T. The closest 16S rRNA gene relatives to strain NE40T are Endozoicomonas montiporae CL-33T (98.2%), Endozoicomonas euniceicola EF212T (97.6%) and Endozoicomonas gorgoniicola PS125T (97.2%). The four strains show genome-wide average nucleotide identity scores above the species level cut-off (95%) with one another and below the cut-off with all Endozoicomonas type strains with publicly available genomes. Digital DNA–DNA hybridization further supported the classification of the strains as a novel species, showing values below 70% when compared with other Endozoicomonas type strains. The DNA G+C content of NE40T was 49.0 mol%, and its genome size was 5.45 Mb. Strain NE40T grows from 15 to 37 °C, with 1–5% (w/v) NaCl, and between pH 6.0 and 8.0 in marine broth and shows optimal growth at 28–32 °C, 2–3% NaCl and pH 7.0–8.0. The predominant cellular fatty acids are summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c), C16 :0 and C14 :0. Strain NE40T presents oxidase, catalase and β-galactosidase activities and can reduce nitrates to nitrites and degrade cellulose, chitin, agarose and xylan. Based on the polyphasic approach employed in this study, we propose the novel species name Endozoicomonas lisbonensis sp. nov. (type strain NE40T=DSM 118084T=UCCCB 212T).