Description of Enterovibrio nigricans sp. nov., reclassification of Vibrio calviensis as Enterovibrio calviensis comb. nov. and emended description of the genus Enterovibrio Thompson et al. 2002 Javier Pascual, 1,2 M. Carmen Macia ´n, 3 David R. Arahal, 2,3 Esperanza Garay 2,3 and Marı ´a J. Pujalte 1,2 Correspondence Marı ´a J. Pujalte maria.j.pujalte@uv.es 1 Instituto Cavanilles de Biodiversidad y Biologı ´a Evolutiva (ICBiBE), Universidad de Valencia, Valencia, Spain 2 Departamento de Microbiologı ´a y Ecologı ´a, Universidad de Valencia, Valencia, Spain 3 Coleccio ´ n Espan ˜ ola de Cultivos Tipo (CECT), Universidad de Valencia, Valencia, Spain Eleven strains of halophilic, facultative anaerobes isolated from healthy and diseased Dentex dentex and Sparus aurata (bony fishes) cultured in Spanish Mediterranean fisheries have been studied by a polyphasic approach that included a wide phenotypic characterization, DNA–DNA hybridization and phylogenetic analysis using 16S rRNA, recA and rpoD gene sequences. All strains were phylogenetically related to Enterovibrio species and Vibrio calviensis. On the basis of sequence analysis and DNA–DNA hybridization data, eight of the strains were identified as Enterovibrio coralii. The remaining three strains formed a tight, independent clade in all sequence analyses and showed less than 70 % DNA–DNA hybridization with strains of the closest Enterovibrio species, from which they could be differentiated by several phenotypic traits. We conclude that these three strains represent a novel species in the genus Enterovibrio and we thus propose the name Enterovibrio nigricans sp. nov., with strain DAl 1-1-5 T (5CECT 7320 T 5CAIM 661 T ) as the type strain. In addition, we propose the reclassification of Vibrio calviensis Denner et al. 2002 as Enterovibrio calviensis comb. nov. (type strain RE35/F12 T 5CIP 107077 T 5DSM 14347 T 5CECT 7414 T ) and we provide an emended description of the genus Enterovibrio. The genus Enterovibrio was proposed by Thompson et al. (2002) to accommodate a group of isolates obtained from larvae of Scophthalmus maximus, described as the novel species Enterovibrio norvegicus. The strains shared some phenotypic characteristics with Vibrio species but were phylogenetically closer to Grimontia hollisae (basonym Vibrio hollisae), although they shared only 94 % 16S rRNA gene sequence similarity. Three years later, a second Enterovibrio species was described, Enterovibrio coralii, isolated from diseased corals (Thompson et al., 2005b). It is also recognized, although no formal proposal has been made, that Vibrio calviensis (Denner et al., 2002) shares greater phylogenetic relatedness with members of Enterovibrio than with Vibrio species, according to recent phylogenetic analyses (Thompson et al., 2005b; Sawabe et al., 2007). In fact, the generic assignment of V. calviensis was already considered somewhat provisional at the time of its description (Denner et al., 2002). During two surveys on bacteria associated with the internal organs of the cultured fish Dentex dentex (Company et al., 1999) and Sparus aurata (Pujalte et al., 2003), a group of strains isolated from the head kidney, unidentifiable by phenotyping, were found to be related to E. coralii by comparative analysis of 16S rRNA gene sequences. On initial isolation, the strains were considered as non- fermentative, so the taxonomic link with the genus Enterovibrio was surprising, and prompted a deeper characterization that has been performed through a wide phenotypic study, DNA–DNA hybridization and phylo- genetic analysis using three housekeeping genes, the 16S rRNA gene, rpoD and recA. Abbreviations: ML, maximum likelihood; MP, maximum parsimony; NJ, neighbour joining. The GenBank/EMBL/DDBJ accession numbers for the rpoD, recA and 16S rRNA gene sequences determined in this study are AM942047– AM942062, AM942063–AM942078 and AM942722–AM942732, respectively, as detailed in Table 1. Photographs of colonies of strain DAl 1-1-5 T , ML and MP trees based on 16S rRNA, recA and rpoD gene sequences and a concatenated dataset, growth responses and cellular fatty acid profiles of individual strains and detailed DNA–DNA hybridization results are available as supplementary material with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2009), 59, 698–704 DOI 10.1099/ijs.0.001990-0 698 001990 G 2009 IUMS Printed in Great Britain