Systematic and Applied Microbiology 38 (2015) 169–175 Contents lists available at ScienceDirect Systematic and Applied Microbiology j ourna l h omepage: www.elsevier.de/syapm Multilocus Sequence Analysis of the redefined clade Scophthalmi in the genus Vibrio Eva Tarazona, Alba Pérez-Catalu ˜ na, Teresa Lucena, David R. Arahal, M. Carmen Macián, María J. Pujalte ∗ Colección Espa˜ nola de Cultivos Tipo (CECT) and Departamento de Microbiología y Ecología, Universitat de València, Spain a r t i c l e i n f o Article history: Received 27 January 2015 Received in revised form 13 March 2015 Accepted 18 March 2015 Keywords: Vibrio scophthalmi Vibrio ichthyoenteri Vibrio ponticus MLSA rpoD recA mreB a b s t r a c t A Multilocus Sequence Analysis (MLSA) was performed on members of the Scophthalmi clade in the genus Vibrio, including type and reference strains of the species V. scophthalmi, V. ichthyoenteri, and 39 strains phenotypically identified as Vibrio ichthyoenteri-like, with the aim of better defining boundaries between these two closely related, fish-associated species. The type strain of V. ponticus, recently added to the clade Scophthalmi, was also included. The study was based on partial sequences of the protein-coding housekeeping genes rpoD, mreB, recA, ftsZ, and gyrB, and the 16S rRNA. While the 16S rRNA gene-based trees were unable to pull apart members of V. scophthalmi or V. ichthyoenteri, both the other individual gene trees and the trees obtained from the five-genes concatenated sequences were able to consistently differentiate four subclades within the main clade, corresponding to the bona fide V. scophthalmi, V. ichthyoenteri, and two small ones that may represent a new species each. The best genes to differentiate V. scophthalmi from V. ichthyoenteri were rpoD, recA, and mreB. Vibrio ponticus failed to associate to the clade in the MLSA and in most single gene trees for which it should not be considered part of it. In this study we also confirm using genomic indexes that V. ichthyoenteri and V. scophthalmi are two separate species. © 2015 Elsevier GmbH. All rights reserved. Introduction More than a hundred species are currently recognized in Vib- rio, the largest genus in the Family Vibrionaceae (http://www. bacterio.net/). The multiple roles that Vibrio species perform in marine habitats, from symbiotic luminescence to pathogenicity in different organisms, justify the interest of developing a sound clas- sification and accurate identification tools for all species. The use of MLSA as a basis for the classification of Vibrio spp. has resulted in the establishment of phylogenetically defined, subgeneric ranks that distribute the numerous Vibrio species in clades, based on their clustering in concatenated nine-gene phylogenetic trees of the Abbreviations: ANI, Average Nucleotide Identity; ATCC, American Type Cul- ture Collection; CECT, Colección Espa ˜ nola de Cultivos Tipo; LMG, Laboratorium voor Microbiologie; UGent, Universiteit Gent; DDH, DNA-DNA hybridization; ML, Maxi- mum Likelihood; MLSA, Multilocus Sequence Analysis; MP, Maximum Parsimony; NCIMB, National Collection of Industrial and Marine Bacteria; NJ, Neighbor Joining; ST, Sequence Types. ∗ Corresponding author at: Departamento de Microbiología y Ecología, Facultad de Biología, Universitat de València, Doctor Moliner, 50, 46100 Burjassot (Valencia) Spain. Tel.: +34 96 354 31 42; fax: +34 96 354 31 87. E-mail address: Maria.J.Pujalte@uv.es (M.J. Pujalte). genus [30,31]. Although Vibrio clades (usually named after the earli- est species contained in them) have no formal correspondence with any prokaryotic taxonomic rank in the Bacteriological Code, the use of this classification has become popular, as it allows to easily pin- point the “neighborhood” of a given species in this already quite extensive genus. Some clades (Harveyi clade, Splendidus clade) contain more than a dozen species but many others only a pair. Most of them cannot be delineated using 16S rRNA sequence com- parisons alone, and less conserved gene sequences are required. In spite of these limitations their pragmatic advantages push forward their use among researchers. Two Vibrio species, Vibrio ichthyoenteri and Vibrio scophthalmi form the clade Scophthalmi [30]. They were described in the late’90s from the intestine of two different flat fishes, the Japanese flounder, Paralichthys olivaceus [12], and turbot, Scophthalmus maximus [6], respectively. V. ichtyoenteri was responsible for the intestinal necrosis of flounder larvae [12,13] and has been isolated as a major component of the intestinal microbiota of Senegalese sole (Solea senegalensis) [20], from kidneys of wild caught spotted rose snapper (Lutjanus guttatus) [9], from the gastrointestinal tract of flounder larvae (P. olivaceus) [10,14,32] and from the digestive tract of larval ballan wrasse (Labrus bergylta) and goldsinny wrasse (Ctenolabrus rupestris) [5]. V. scophthalmi, considered a possible http://dx.doi.org/10.1016/j.syapm.2015.03.005 0723-2020/© 2015 Elsevier GmbH. All rights reserved.