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1 Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA
2 The Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Launceston, Tasmania, Australia
Correspondence
Paul V. Dunlap
pvdunlap{at}umich.edu
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97.4 % among members of the V. fischeri group, but were 
95.5 % for members of this group in comparison with type species of other genera of the Vibrionaceae (i.e. Photobacterium and Vibrio, with which they overlap in G+C content, and Enterovibrio, Grimontia and Salinivibrio, with which they do not overlap in G+C content). Combined analysis of the recA, rpoA, pyrH, gyrB and 16S rRNA gene sequences revealed that the species of the V. fischeri group form a tightly clustered clade, distinct from these other genera. Furthermore, phenotypic traits differentiated the V. fischeri group from other genera of the Vibrionaceae, and a panel of 13 biochemical tests discriminated members of the V. fischeri group from type strains of Photobacterium and Vibrio. These results indicate that the four species of the V. fischeri group represent a lineage within the Vibrionaceae that is distinct from other genera. We therefore propose their reclassification in a new genus, Aliivibrio gen. nov. Aliivibrio is composed of four species: Aliivibrio fischeri comb. nov. (the type species) (type strain ATCC 7744T =CAIM 329T =CCUG 13450T =CIP 103206T =DSM 507T =LMG 4414T =NCIMB 1281T), Aliivibrio logei comb. nov. (type strain ATCC 29985T =CCUG 20283T =CIP 104991T =NCIMB 2252T), Aliivibrio salmonicida comb. nov. (type strain ATCC 43839T =CIP 103166T =LMG 14010T =NCIMB 2262T) and Aliivibrio wodanis comb. nov. (type strain ATCC BAA-104T =NCIMB 13582T =LMG 24053T).
GenBank accession numbers for 16S rRNA gene sequences and a table with complete phenotyping data are available as supplementary material with the online version of this paper.
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). Members of this family are relatively easy to culture and common in marine environments, and several species are important animal pathogens. Understanding the ecology and evolution of the Vibrionaceae requires a taxonomy that accurately reflects intrafamilial species relationships; however, a number of groups in the Vibrionaceae remain indefinitely resolved (Farmer et al., 2005). One of these groups is the Vibrio fischeri group, which contains four species, Vibrio fischeri (Beijerinck, 1889), Vibrio logei (Harwood et al., 1980), Vibrio salmonicida (Egidius et al., 1986) and Vibrio wodanis (Lunder et al., 2000). Previous taxonomic studies have shown that these four species are closely related to each other, but there is disagreement about the genus into which these species should be classified (Thyssen & Ollevier, 2005). Some analyses of V. fischeri, for example, have identified phenotypic traits (Reichelt & Baumann, 1973) or molecular characters (Baumann & Baumann, 1977) consistent with membership in Photobacterium, whereas other studies have placed V. fischeri in Vibrio based on other phenotypic traits (Hendrie et al., 1970) or on polyphasic analysis of phenotypic and molecular characteristics (Baumann et al., 1980). Recently, Thompson et al. (2005) suggested that the Vibrionaceae should be divided into four families, with the placement of V. fischeri, V. logei and V. wodanis in an unnamed monophyletic group, sister to the Vibrionaceae (which the authors suggest should consist only of genus Vibrio) and separate from the family ‘Photobacteriaceae’. The ambiguity of the taxonomy and phylogenetic placement of the V. fischeri group is reflected in current species naming. Photobacterium fischeri and Photobacterium logei are synonyms of V. fischeri and V. logei, although the two species have characteristics more similar to those of V. wodanis and V. salmonicida than to those of Photobacterium phosphoreum, the type species of Photobacterium, whereas neither V. wodanis nor V. salmonicida has been considered to be a member of Photobacterium. The conflicting concepts regarding classification of the V. fischeri group indicate the need to resolve the relationship of this group relative to other species in the Vibrionaceae. The importance of a more definitive resolution of groups currently classified in Vibrio is especially evident with the advent of genome sequence analysis and subsequent attempts to interpret differences between pathogenic and non-pathogenic species in a systematic context (e.g. Ruby et al., 2005). In this study, we tested the ability of genetic loci and phenotypic traits to distinguish members of the V. fischeri group from representatives of Vibrio, Photobacterium and other genera in the family. Based on the results, which demonstrate that the V. fischeri group is distinct phylogenetically and phenotypically from other genera in the Vibrionaceae, we propose the establishment of a new genus, Aliivibrio gen. nov., for the members of the V. fischeri group.
First, we determined the extent of 16S rRNA gene sequence identity between members of the V. fischeri group and representative species of Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio. Despite the importance of the 16S rRNA gene for bacterial taxonomy, special care is required for its use in taxonomy of the Vibrionaceae. Several loop regions of the 16S rRNA gene have highly variable sequences and are difficult to align objectively; at the same time, stem regions are sometimes invariant between different species. To minimize difficulties associated with 16S rRNA gene sequence comparison, we used direct optimization (Wheeler, 1996) for alignment of sequences, as implemented by the program POY (version 4.0 beta build 1822) (Varón et al., 2007). The direct optimization method iteratively evaluates alignments in a phylogenetic context, and this method of analysis results in a more rigorously tested alignment; it is therefore an improvement over other alignment programs and is especially useful for sequences like those of 16S rRNA genes in which insertion–deletion events are common and result in length differences among homologous sequences. For the analysis, we compared 16S rRNA gene sequences of 92 type strains from the Vibrionaceae, including the type strains of the four species of the V. fischeri group. Accession numbers of the 16S rRNA gene sequences used here can be found in Supplementary Table S1, available in IJSEM Online. For direct optimization, gap and nucleotide change costs were set to 1 [using the command transform((all, tcm:(1,1)))]. Ten initial trees were built using random addition sequence [build()]. Each starting tree was subjected to branch swapping, alternating subtree pruning-regrafting and tree bisection-reconnection [swap()]. After 20 replicates, the shortest three trees were kept [select(best:3)] and submitted to tree fusing [fuse()] and to 100 iterations of parsimony ratcheting, with reweighting 20 % of the characters [transform((all, static_approx))] by 5 and keeping up to five trees [perturb(iterations:100, ratchet:(0.2,5), swap(trees:5))]. Tree fusing and ratcheting independently found the same shortest phylogenetic hypothesis and alignment, with an alignment length of 1397 characters (the resulting alignment and tree are available from the authors on request). After the alignment was constructed, pairwise distances between 16S rRNA gene sequences were calculated in PAUP* (Swofford, 2003) using the Kimura two-parameter model.
High sequence identities, of 97.4 % or more, were obtained for the 16S rRNA genes of the V. fischeri group (Table 1), indicating a close relationship among these strains. In contrast, 16S rRNA gene sequence identities between type strains of species in the V. fischeri group and other species of the Vibrionaceae were consistently lower. Specifically, sequence identities for V. fischeri ATCC 7744T and P. phosphoreum ATCC 11040T (the type species of Photobacterium) and Vibrio cholerae ATCC 14035T (the type species of Vibrio) are 95.4 and 94.6 %, respectively. The 16S rRNA gene sequence identities of V. fischeri ATCC 7744T to type strains of type species of other genera in the family were below 92.3 %. These results indicate that, based on 16S rRNA gene sequence identities, the four species of the V. fischeri group form a group within the Vibrionaceae that is distinct from other species in the family.
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for Vibrionaceae phylogeny, together with gyrB, the sequence of which discriminates species in the Vibrionaceae (Ast & Dunlap, 2005), and sequences of the 16S rRNA gene. These genes were analysed simultaneously using type strains of 43 representative species of the Vibrionaceae, including representatives of the five well-characterized genera in the family. Because inclusion of additional taxa improves phylogenetic analysis (Graybeal, 1998; Hillis, 1998), we also included sequence data for V. fischeri strains ES114 (from the light organ of the sepiolid squid Euprymna scolopes; Ruby et al., 2005), MJ-1 (from the light organ of the monocentrid fish Monocentris japonicus; Ruby & Nealson, 1976), etasm.1.1 (from the light organ of the sepiolid squid Euprymna tasmanica; this study) and lpeal.1.1 (from the accessory nidamental gland of the loliginid squid Loligo pealei; this study) and for V. salmonicida strain LFI1238 (http://www.sanger.ac.uk/Projects/V_salmonicida/). As outgroup in the analysis, we used Photorhabdus luminescens subsp. laumondii TT01T (Duchaud et al., 2003), giving a total of 49 strains. PCR amplifications were done as described previously (Thompson et al., 2005; Ast & Dunlap, 2005) except for pyrH, for which primer PBPRA2966Rv (5'-GAATCGGCATTTTATGGTCACG-3') was used instead of pyrH-02-R. Sequencing was done by staff of the University of Michigan Sequencing Core using dye-terminator cycle sequencing on a Perkin-Elmer ABI 3730 or 3700 DNA Analyzer. Gene sequences from the five loci were concatenated and used for direct optimization analysis. Gaps and nucleotide changes were set to 1 for the 16S rRNA gene, as they were in the analysis of percentage sequence identity of the 16S rRNA gene. For the four protein-coding genes, initial gap costs were set to 2, with extension gaps and nucleotide change costs set to 1. Analysis proceeded as described above for the 16S rRNA gene (except for the use of 35 replicates of builds and swapping instead of the 20 replicates used in the 16S rRNA gene analysis and 20 % of fragments, instead of characters, were reweighted in the ratchet). A single, shortest hypothesis was found after fusing and ratcheting (see legend to Fig. 1 for tree statistics). Confidence values were obtained with 10 000 jackknife resampling replicates (Farris et al., 1996) using TNT (Goloboff et al., 2005).
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). The V. fischeri group forms a clade closely related to Photobacterium and Vibrio but clearly separate from both these genera and more distantly related to other genera of the Vibrionaceae. We note parenthetically here also that these relationships accord with the overlap in G+C contents between the V. fischeri group and Photobacterium and Vibrio and the lack of overlap in these values between the V. fischeri group and Enterovibrio, Grimontia and Salinivibrio (see Table 2). These results, which are consistent with the percentage identities in 16S rRNA gene sequences (Table 1), indicate that the members of the V. fischeri group form a phylogenetic lineage of the Vibrionaceae that is distinct from Photobacterium, Vibrio and other genera in the family.
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; see references cited in Supplementary Table S2 for test methods), was used. Several of these tests were found to distinguish the V. fischeri group from other genera in the Vibrionaceae (Table 2). Furthermore, a panel of 13 tests was identified that differentiated species within the V. fischeri group from each other and from species of Photobacterium and Vibrio, the two genera most closely related to the V. fischeri group (Table 3). For this species-level analysis, strains used in the multilocus phylogenetic analysis (Fig. 1), together with type strains of six Photobacterium species and 32 Vibrio species and additional strains of V. fischeri, were examined. Discriminatory tests were identified using the GBEST algorithm, part of the PIBWin suite of tools for probabilistic identification of bacteria (Bryant, 1991, 2004). All taxa could be differentiated by at least two tests with a test difference 70 % (Willcox et al., 1973) (Table 3; complete results for all 46 tests can be found in Supplementary Table S2). The results of these phenotypic comparisons are consistent with the differences revealed by the 16S rRNA gene sequence identity comparison and by the multilocus phylogenetic analysis of the recA, rpoA, pyrH, gyrB and 16S rRNA genes.
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Description of Aliivibrio gen. nov.
Aliivibrio (A.li.i.vib'ri.o. L. n. alius other, another; N.L. masc. n. Vibrio a bacterial genus name; N.L. masc. n. Aliivibrio the other Vibrio).
Gram-negative, motile, rod-shaped cells with one or more sheathed flagella. Conforms to the description of the family Vibrionaceae. Some strains are luminous. Oxidase-positive, fermentative and can utilize glucose as a sole carbon source; sensitive to the vibriostatic agent O/129 at 10 µg. Grow on media with 1 % (w/v) NaCl but not with 10 % (w/v) NaCl. Species are arginine dihydrolase-negative, do not hydrolyse gelatin, do not form acetoin (Voges–Proskauer test) and are sensitive to novobiocin at 5 µg. Aliivibrio fischeri ferments gentiobiose and is urease-positive; all other species of the genus are negative for these two characteristics. All species utilize acetate as a sole carbon source except A. fischeri. Species other than A. salmonicida have yellow–orange cell-associated pigment. DNA G+C content is between 38 and 42 mol%. Found in the marine environment, often associated with animals; some species are mutualistic symbionts or pathogens of marine animals. Member of the Gammaproteobacteria. The type species is Aliivibrio fischeri.
Description of Aliivibrio fischeri (Beijerinck 1889) comb. nov.
Basonym: Vibrio fischeri (Beijerinck 1889) Lehmann and Neumann 1896.
Other synonym: Photobacterium fischeri (Beijerinck 1889) Reichelt and Baumann 1973.
The description is the same as that given for Photobacterium fischeri by Reichelt & Baumann (1973) with the following additions. Negative for the Voges–Proskauer (acetoin) test, indole production, gelatinase and agarolysis. Urease-positive. Resistant to carbenicillin (100 µg) and ampicillin (10 µg). The type strain is ATCC 7744T =CAIM 329T =CCUG 13450T =CIP 103206T =DSM 507T =LMG 4414T =NCIMB 1281T.
Description of Aliivibrio logei (Harwood et al. 1980) comb. nov.
Basonym: Photobacterium logei (ex Bang et al. 1978) Harwood et al. 1980.
Other synonym: Vibrio logei (Harwood et al. 1980) Baumann et al. 1981.
The description is the same as that given for Photobacterium logei by Bang et al. (1978) with the following additions. Indole-negative. Resistant to carbenicillin (100 µg) and ampicillin (10 µg) and sensitive to novobiocin (5 µg). The type strain is ATCC 29985T =CCUG 20283T =CIP 104991T =NCIMB 2252T.
Description of Aliivibrio salmonicida (Egidius et al. 1986) comb. nov.
Basonym: Vibrio salmonicida Egidius et al. 1986.
The description is the same as that given for Vibrio salmonicida by Egidius et al. (1986) with the following additions. Negative for indole production and agarolysis. Resistant to carbenicillin (100 µg) and ampicillin (10 µg) and sensitive to novobiocin (5 µg). The type strain is ATCC 43839T =CIP 103166T =LMG 14010T =NCIMB 2262T.
Description of Aliivibrio wodanis (Lunder et al. 2000) comb. nov
Basonym: Vibrio wodanis Lunder et al. 2000.
The description is the same as that given for Vibrio wodanis by Lunder et al. (2000) with the following additions. Sensitive to carbenicillin (100 µg) and novobiocin (5 µg). The type strain is ATCC BAA-104T =NCIMB 13582T =LMG 24053T.
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ACKNOWLEDGEMENTS |
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-D-galactosidase (4-nitrophenyl 
-glutamyl transpeptidase (L-glutamic acid 5-(4-nitroanilide); 6, aesculin hydrolysis; 7,