Comparative genomic analyses identify the Vibrio harveyi genome sequenced strains BAA-1116 and HY01 as Vibrio campbellii Baochuan Lin, 1 Zheng Wang, 1 Anthony P. Malanoski, 1 Elizabeth A. O’Grady, 2 Charles F. Wimpee, 2 Varaporn Vuddhakul, 3 Nelson Alves Jr, 4 Fabiano L. Thompson, 4 Bruno Gomez-Gil 5 and Gary J. Vora 1 * 1 Center for Bio/Molecular Science & Engineering, Naval Research Laboratory, Washington, DC, USA. 2 Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, USA. 3 Department of Microbiology, Prince of Songkla University, Hat Yai, Thailand. 4 Department of Genetics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. 5 CIAD, A.C., Mazatlán Unit for Aquaculture, Sinaloa, Mexico. Summary Three notable members of the Harveyi clade, Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyti- cus, are best known as marine pathogens of commer- cial and medical import. In spite of this fact, the discrimination of Harveyi clade members remains dif- ficult due to genetic and phenotypic similarities, and this has led to misidentifications and inaccurate esti- mations of a species’ involvement in certain environ- ments. To begin to understand the underlying genetics that complicate species level discrimination, we com- pared the genomes of Harveyi clade members isolated from different environments (seawater, shrimp, corals, oysters, finfish, humans) using microarray-based comparative genomic hybridization (CGH) and multilo- cus sequence analyses (MLSA). Surprisingly, we found that the only two V. harveyi strains that have had their genomes sequenced (strains BAA-1116 and HY01) have themselves been misidentified. Instead of belonging to the species harveyi, they are actually members of the species campbellii. In total, 28% of the strains tested were found to be misidentified and 42% of these appear to comprise a novel species. Taken together, our findings correct a number of species misidentifications while validating the ability of both CGH and MLSA to distinguish closely related members of the Harveyi clade. Introduction The eight Vibrio species currently recognized as members of the Harveyi clade (V. harveyi, V. campbellii, V. alginolyti- cus, V. rotiferianus, V. parahaemolyticus, V. natrigens, V. mytili and V. azureus) (Sawabe et al., 2007; Yoshizawa et al., 2009) are a subset of the Vibrio core group (Reichelt et al., 1976; Dorsch et al., 1992). Members of this clade are commonly found in marine and estuarine surface waters and sediments, as commensals on the surface or within the intestinal flora of marine animals, as opportunistic patho- gens, or as primary pathogens of many commercially farmed marine invertebrate and vertebrate species (O’Brien and Sizemore, 1979; Thompson et al., 2004). In addition to thriving in similar environments, members of the Harveyi clade also share a high degree of genetic and phenotypic similarity; so much so that traditional pheno- typic identification methods are often unable to confidently identify and differentiate these sister species (Sawabe et al., 2007; Cano-Gomez et al., 2009). For example, V. harveyi, V. campbellii and V. rotiferianus, which form the most recent subclade of speciation within the Harveyi clade (Pascual et al., 2009), have nearly indistinguishable phenotypes (Bryant et al., 1986; Gomez-Gil et al., 2004). These similarities have confounded typing schemes and resulted in documented misidentifications (Gauger and Gomez-Chiarri, 2002; Gomez-Gil et al., 2004). While not exceedingly problematic, these misidentifications do have the potential to overemphasize the importance of a species in a particular setting, especially since most misidentifica- tions are initially characterized as V. harveyi. Considering the economic importance and seemingly continually expanding host range of the Harveyi clade (Austin et al., 2005; Rosenberg et al., 2007; Cervino et al., 2008; Defoirdt et al., 2008), there remains a contin- ued interest in the development of methods to identify and differentiate its members. In contrast with phenotypic Received 20 August, 2009; accepted 7 October, 2009. *For corre- spondence. E-mail gvora@cbmse.nrl.navy.mil; Tel. (+1) 202 767 0394; Fax (+1) 202 404 8688. Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://www3.interscience.wiley.com/ authorresources/onlineopen.html Environmental Microbiology Reports (2010) 2(1), 81–89 doi:10.1111/j.1758-2229.2009.00100.x © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd