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Mutation Is the Main Driving Force in the Diversification of the Vibrio Splendidus Clade

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Mutation Is the Main Driving Force in the Diversification of the Vibrio Splendidus Clade Microbes Environ. Vol. 24, No. 4, 281–285, 2009 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME09128 Mutation is the Main Driving Force in the Diversification of the Vibrio splendidus Clade TOMOO SAWABE1*, SHINICHI KOIZUMI1, YOUHEI FUKUI1, SATOSHI NAKAGAWA1, ELENA P. IVANOVA2,3, KUMIKO KITA-TSUKAMOTO4, KAZUHIRO KOGURE4, and FABIANO L. THOMPSON5 1Laboratory of Microbiology, Faculty of Fisheries Sciences, Hokkaido University, 3–1–1 Minato-cho, Hakodate 041–8611, Japan; 2Swinburne University of Technology, PO Box 218, Hawthorn, Victoria 3122, Australia; 3Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Vladivostok 690022, Pr. 100 Let Vladivostoku 159, Russian Federation; 4Ocean Research Institute, University of Tokyo, 1–15 Minamindai, Nakano-ku, Tokyo 164–8639, Japan; and 5Department of Genetics, Center of Health Sciences, Federal University of Rio de Janeiro, Ilha do Fundão, Caixa Postal 68011, CEP 21944–970, RJ, Brazil (Received May 18, 2009—Accepted July 30, 2009—Published online September 3, 2009) The Vibrio splendidus clade is the biggest in Vibrionales composed of 11 described species (25). Diversification of these species may have occurred 260 million years ago. The main driving forces of speciation in this clade have never been studied. Population biological parameters (population base recombination rate (ρ), population base mutation rate (θ), and index of association (Ia)) were determined among 16 strains of 9 defined species in the Splendidus cluster. A comparison of individual gene phylogeny indicated significant incongruence in tree topology, which suggests the occurrence of recombination between species. Homologous recombination between species was detected at four loci. However, the mutation rate θ was higher than the recombination rate ρ, suggesting that mutation is the main driving force in the diversification of V. splendidus-related species. Key words: diversification, population biology, vibrios, Vibrio splendidus, multilocus sequence analysis Vibrio splendidus was originally described by Beijerinck 99.8% average amino acid identity at gene loci, but a rather in 1900 as a luminous marine bacterium (1, 2, 22). The taxo- broad GC content. The high genomic similarity might sug- nomic status of this species was confirmed through DNA- gest that these species readily exchange DNA at conserved DNA hybridization by Reichelt et al. (22). V. splendidus- loci. To test this hypothesis, we analyzed the relative contri- related strains isolated from marine environments remained bution of mutation and recombination to the diversification in focus due to the psychrophilic nature of these vibrios in using an extended set of strains belonging to the Splendidus the global warming era (27, 34, 35). A role for V. splendidus- group by means of MLSA. related strains in the mortality of mollusks and fish was also reported (6, 9, 13). V. splendidus and related species are Materials and Methods widespread in marine environments (14, 30, 32, 34, 35). Recently, two genetically distinctive populations of V. splen- Bacterial strains didus-related strains from North Atlantic coastal water have Four strains of V. cyclitrophicus, three strains of V. kanaloae, been distinguished. Bacteria of these two populations dif- two strains of V. lentus, two strains of V. gigantis, and one strain each of V. chagasii, V. crassostreae, V. fortis, V. pelagius, V. splen- fered in range of temperature for growth and had less than didus, and V. tasmaniensis were used (Figs. 1 and 2). The V. 1% of the 16S rRNA gene sequence heterogeneity (30, 32). cyclitrophicus strains showed major phenotypic heterogeneity in the Because V. splendidus-related species share niches in the production of brown soluble pigment (LMG 21439, LMG 23440 environment, including the gut and tissue of bivalves, it and LMG 7905), requirement of organic growth factor, and growth would be reasonable to assume that horizontal gene transfer on ZoBell2216E medium (LMG 7905). The poor-growing V. and recombination are important forces leading to diver- cyclitrophicus LMG 7905 was deposited as Vibrio ‘algosus’ (39). These four strains were confirmed to be conspecific by reciprocal sification in this group. The multilocus sequence analysis DNA-DNA hybridization experiments showing 72.2% to 100% (MLSA) is a reliable tool for both inferring the taxonomic values. backbone of species groups and estimating the relative con- tributions of mutation and recombination (31, 33, 36). Our Sequencing of the protein coding genes recent MLSA clearly separated the V. splendidus clade from Bacterial DNA of all strains was prepared for PCR amplification all other recognized species of vibrios (25). However, V. and sequencing with Wizard DNA purification system (Promega, Madison, WI, USA). The DNA (100 ng) was used as a template in a splendidus-related species are genetically and phenotypically PCR to amplify four protein-encoding genes [gapA (glyceraldehyde similar (29). Overall, the species have more than 30% 3-phosphate dehydrogenase gene), mreB (actin-like cytoskeleton reciprocal DNA-DNA similarity, 90.6–96.5% nucleotide gene), ftsZ (cell division protein gene), and topA (topoisomerase I sequence similarity of MLSA concatenated genes and 96.5– gene)] for the multi-locus sequence analysis (25). The PCR prod- ucts were analyzed on a 1.5% agarose gel with a molecular weight * Corresponding author. E-mail: [email protected]; Tel: standard for quantification of the PCR yield. The products which +81–138–40–5569; Fax: +81–138–40–5569. produced a single band on the agarose gel were purified using a Gel 282 SAWABE et al. Fig. 1. Unrooted phylogenetic trees based on house keeping genes. (A) gapA, (B) mreB, (C) ftsZ and (D) topA. This figure combines the results of three analyses i.e., neighbor-joining, maximum parsimony and maximum likelihood. The topology shown was obtained by neighbor-joining, and values (%) below branches are the results of a bootstrap analysis using 100 replications. The branches indicated by bootstrap values also supported by the most parsimonious tree and in the maximum likelihood analysis (p<0.01). and PCR purification system (Promega). DNA sequencing was Detection of recombination conducted by the Shimadzu Biotech sequencing service using a Incongruence of the topology of each tree may reflect conflicting RISA384 sequencer (Kyoto, Japan). phylogenetic signals. We conducted a multi-step analysis to evalu- The sequences were aligned using the ClustalX program (28). ate probable recombination signals among V. splendidus-related Domains used to construct the phylogenetic tree shown in Fig. 1 strains. Discordance between sequence data from different loci was were regions of gapA, mreB, ftsZ, and topA sequences available for evaluated by means of the incongruence length difference test (ILD) all species (ca. 80 species) belonging to Vibrionales: positions 204– implemented in PAUP 4b10 (5). A split decomposition analysis was 894, 129–716, 387–976 and 418–1073 (numbering based on the E. conducted using SplitsTree version 4 (10). Using aligned data of the coli K12 sequence, NC000913), respectively. The 161–1364 region gapA (687 bp), ftsZ (507 bp), mreB (567 bp), and topA (656 bp) was used for the phylogenetic analysis based on the 16S rRNA gene genes, a concatenated split network was generated. Furthermore, (Fig. S1). In the final trees, sequences of the nine closest species of probable recombination break points were estimated based on the V. splendidus were retained (Fig. 1). Phylogenetic analyses were MaxChi algorithm (16) using Recombination Detection Program performed using three methods, neighbor-joining (NJ) method (24), (RDP) version 2.0 (15). maximum-likelihood (ML) (7) (Phylip package) and maximum-par- simony (MP) (8) (MEGA ver3.0; close-neighbor-interchange with Population biological analysis sear 1 option). The robustness of each topology was checked using Aligned sequence data were assigned by a non-redundant data- the neighbor-joining method and 100 bootstrap replications. Trees base program, and linkage disequilibrium (including an index of were drawn using MEGA version 3.0 (12). association: Ia) (17) was determined by a web-based program, both of which are available at http://linux.mlst.net/link_dis/index.htm. Diversification of V. splendidus Species Clade 283 Using the same aligned data, ρ and θ were calculated with LDhat (strain number 518) in ZoBell’s study mainly focused on version 2.0, and corrected for haploid organisms. A pairwise calcu- morphology, growth and biochemical characteristics. An lation of radiation time among species in the Splendidus clade was association with marine kelp attributed to the nomenclature conducted based on the ds value of each gene using MEGA4.0 (12, of the species. The strain V. ‘algosus’ is maintained in sev- 25). Based on matrices normalized to E. coli-Salmonella radiation as an event 100 million years ago (11, 23), a UPGMA tree was eral culture collections under the following strain num- reconstructed. bers: LMG 7905=ATCC 14390=NCIMB 2073. Nevertheless, the species has not been validly described and the name GenBank accession numbers V. ‘algosus’ has not been included in the Approved List of The GenBank accession numbers for 16S rRNA gene sequences Bacterial Names (26). Even though V. ‘algosus’ has been of V. cyclitrophicus strains are DQ481607 to DQ481610; gapA described, it does not have a valid taxonomic standing: gene sequences are DQ481614 to DQ481616, DQ481618, DQ914235, DQ981851,
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