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Yet most knowledge of the genus comes from human infection-associated lineages with genomes swollen by the recent accretion of foreign DNA conveyed by mobile elements. Because invertebrates dominate terrestrial animal diversity and biomass, they would be predicted to constitute a major but little-explored reservoir of enterococcal diversity. We therefore systematically examined\n                      <jats:italic toggle=\"yes\">Enterococcus<\/jats:italic>\n                      association and species diversification in invertebrate hosts of the comparatively natural, isolated, but well-characterized environment of the Azorean island of Terceira. Over 100 invertebrate specimens were examined for associated enterococci, which were taxonomically classified by whole-genome sequencing. Supporting the existence of a large pool of uncharacterized enterococci and\n                      <jats:italic toggle=\"yes\">Enterococcus<\/jats:italic>\n                      -adapted genes, 40% (eight of 20) of the\n                      <jats:italic toggle=\"yes\">Enterococcus<\/jats:italic>\n                      species identified were either undescribed, including four candidate new species described here, or very recently discovered. In contrast, control isolates from vertebrates were exclusively of known species typical of sampling elsewhere, discounting geographic isolation as a main driver of the novelty observed. Further, because of the abundance of\n                      <jats:italic toggle=\"yes\">E. casseliflavus<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">E. flavescens<\/jats:italic>\n                      in this collection, we obtained the resolution necessary to quantify the divergence and decipher the drivers of speciation in the controversial division between these naturally vancomycin-resistant species. These findings provide robust support for the existence of a large pool of new species and unexplored adaptive traits in invertebrate-associated enterococci\u2014diverse environmental survival traits optimized for expression in an enterococcal background, and well positioned for transmission into human-associated enterococcal strains.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Enterococci are auxotrophic gut-associated bacteria that co-evolved with their terrestrial hosts over many eons. In the last 75 years\u2014the \u201cantibiotic era\u201d\n                        <jats:italic toggle=\"yes\">\u2014E. faecalis<\/jats:italic>\n                        and\n                        <jats:italic toggle=\"yes\">E. faecium<\/jats:italic>\n                        gained genes for antibiotic resistance and enhanced virulence, emerging as leading causes of multidrug-resistant infection. Little is known about the source of those genes or the pathway by which they entered human-associated strains. A recent global survey suggested a potentially large repository of uncharacterized genetic diversity in the enterococci of invertebrates. We directly tested this prospect by examining enterococci of invertebrate hosts in a largely natural and pastoral environment. Our findings provide clear evidence that invertebrates naturally harbor vast unexplored enterococcal diversity. Moreover, associations are likely driven by intrinsic host selection factors rather than geographic isolation. This expands our knowledge of\n                        <jats:italic toggle=\"yes\">Enterococcus<\/jats:italic>\n                        biodiversity, including the identification of four novel species, identifying a vast reservoir of enterococcal genes available to species that colonize and infect humans.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.01234-26","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T15:24:52Z","timestamp":1787239492000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Novelty, diversity, and genetic dark matter in enterococci of invertebrates"],"prefix":"10.1128","volume":"17","author":[{"given":"Bruna F.","family":"Sgardioli","sequence":"first","affiliation":[{"name":"Faculty of Agricultural and Environmental Sciences, University of the Azores","place":["Angra do 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