Systematic and Applied Microbiology 36 (2013) 96–100 Contents lists available at SciVerse ScienceDirect Systematic and Applied Microbiology j ourna l ho mepage: www.elsevier.de/syapm Vibrio toranzoniae sp. nov., a new member of the Splendidus clade in the genus Vibrio Aide Lasa, Ana L. Diéguez, Jesús L. Romalde ∗ Departamento de Microbiología y Parasitología, CIBUS, Universidad de Santiago de Compostela, Campus Sur s/n, 15782 Santiago de Compostela, Spain a r t i c l e i n f o Article history: Received 22 August 2012 Received in revised form 19 November 2012 Accepted 27 November 2012 Keywords: Vibrio toranzoniae sp. nov. Splendidus clade MLSA DNA–DNA hybridization a b s t r a c t Four motile facultative anaerobic marine isolates (Vb 10.8 T [ CECT 7225 T , CAIM 1869 T ], CMJ 9.4 [ CECT 8091, CAIM 1870], CMJ 9.11 and Cmf 13.9), were obtained from cultured clams (Venerupis philippinarum and Venerupis decussata) in Galicia (NW Spain). These isolates were studied by a polyphasic approach, including a phylogenetic analysis based on sequences of 16S rRNA and five housekeeping genes atpA, recA, pyrH, rpoA and rpoD, that supported their inclusion in the Splendidus clade of the genus Vibrio, forming a well-defined group separated from the others species of the clade. DNA–DNA hybridizations with the type strains of species showing more than 98.5% 16S rRNA gene sequence similarity rendered values of hybridization below 60%. These isolates could be differentiated from the closest relatives on the basis of several phenotypic and chemotaxonomic features. These results demonstr8ated that the strains constitute a novel specie of the genus for which the name Vibrio toranzoniae sp. nov. is proposed, with Vb 10.8 T (=CECT 7225 T CAIM 1869 T ) as the type strain. © 2012 Elsevier GmbH. All rights reserved. Vibrios are widely distributed in aquatic environments, includ- ing estuary and marine coastal waters and sediments, where they are frequently associated with eukaryotic organisms. The increasing number of environmental studies has shown the great abundance of vibrios in these environments and the high diver- sity among members of the family Vibrionaceae [26]. Moreover the development of molecular techniques such as multilocus sequence analysis (MLSA), DNA–DNA hybridization and amplified fragment length polymorphism (AFLP), contributed to the descrip- tion of novel species of the genus Vibrio in recent years [5,19], being nowadays 94 species described within the genus Vibrio (www.vibriobiology.net). These molecular techniques also allowed the classification of Vibrio species into 14 clades, being the largest the Splendidus clade with 13 species described to date, including Vibrio atlanticus, Vibrio artabrorum, Vibrio celticus, Vibrio chagasii, Vibrio crassostreae, Vibrio cyclitrophicus, Vibrio gallaecicus, Vibrio gigantis, Vibrio kanaloae, Vibrio lentus, Vibrio pomeroyi, Vibrio splen- didus, and Vibrio tasmaniensis [3,4,7,8,10,12,14,27,28]. However, their discrimination exclusively on the basis of biochemical tests is difficult. The species of this clade are the dominant Vibrio species in coastal marine sediments, seawater and bivalves in temperate climates [11,20]. In addition, some of these species have been asso- ciated with mortalities of a wide range of marine animals such as molluscs, crustacean and fish [3]. ∗ Corresponding author. Tel.: +34 881 816908; fax: +34 881 816966. E-mail address: jesus.romalde@usc.es (J.L. Romalde). In a previous study on the diversity of vibrios associated with reared clams in Galicia (NW Spain), a collection of isolates was analysed by AFLP. As a result of this analysis, a group of four strains (cluster 26) could not be assigned to any of the currently known species of the genus Vibrio [2]. In this study, we report on the taxonomic characterization of these four strains obtained from Manila (Venerupis phillipinarum) and carpet shell (Venerupis decussata) clams cultured in Galicia. Bacterial isolates [Vb 10.8 T ( CECT 7225 T , CAIM 1869 T ), CMJ 9.4 ( CECT 8091, CAIM 1870), CMJ 9.11 and Cmf 13.9] were obtained from cultured clams on the Galician coast (NW Spain) as previously described [2]. Strain Vb 10.8 was isolated from Ría de Vigo (42 ◦ 20 ′ 50 ′′ N/8 ◦ 38 ′ 12 ′′ W) in December 2004 and the strains CMJ 9.4, CMJ 9.11 and Cmf 13.9 were isolated from Ría de Camari ˜ nas (43 ◦ 7 ′ 57 ′′ N/9 ◦ 11 ′ 40 ′′ W) in January 2005. The type strains V. atlanticus CECT 7223 T , V. artabrorum CECT 7226 T , V. celti- cus CECT 7224 T , V. chagasii LMG 21353 T , V. crassotreae LMG 20537 T , V. cyclitrophicus LMG 21359 T , V. gallaecicus CECT 7244 T , V. gigan- tis LMG 22741 T , V. kanaloae LMG 20539 T , V. lentus CECT 5110 T , V. pomeroyi LMG 20537 T , V. splendidus CECT 628 T and V. tasmanien- sis LMG 20012 T were obtained from the CECT (Spanish Collection of Type Cultures), BCCM/LMG Bacteria collection (Belgian Coor- dinated Collections of Microorganisms) and from our laboratory collection, and included in the study for taxonomic comparison. All strains were cultured on marine agar (MA, Pronadisa) at 25 ◦ C for 24 h. Cultures were maintained frozen at -80 ◦ C in marine broth (MB, Pronadisa) supplemented with 15% (v/v) glycerol. The strains were subjected to the following phenotypic tests [3,15]: Gram stain, oxidase, catalase, cell morphology and motility, 0723-2020/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.syapm.2012.11.005