Multilocus Sequence Analysis for the Assessment of Phylogenetic Diversity and Biogeography in <i>Hyphomonas</i> Bacteria from Diverse Marine Environments

<div><p><i>Hyphomonas</i>, a genus of budding, prosthecate bacteria, are primarily found in the marine environment. Seven type strains, and 35 strains from our collections of <i>Hyphomonas</i>, isolated from the Pacific Ocean, Atlantic Ocean, Arctic Ocean, South China Sea and the Baltic Sea, were investigated in this study using multilocus sequence analysis (MLSA). The phylogenetic structure of these bacteria was evaluated using the 16S rRNA gene, and five housekeeping genes (<i>leuA</i>, <i>clpA</i>, <i>pyrH</i>, <i>gatA</i> and <i>rpoD</i>) as well as their concatenated sequences. Our results showed that each housekeeping gene and the concatenated gene sequence all yield a higher taxonomic resolution than the 16S rRNA gene. The 42 strains assorted into 12 groups. Each group represents an independent species, which was confirmed by virtual DNA-DNA hybridization (DDH) estimated from draft genome sequences. <i>Hyphomonas</i> MLSA interspecies and intraspecies boundaries ranged from 93.3% to 96.3%, similarity calculated using a combined DDH and MLSA approach. Furthermore, six novel species (groups I, II, III, IV, V and XII) of the genus <i>Hyphomonas</i> exist, based on sequence similarities of the MLSA and DDH values. Additionally, we propose that the <i>leuA</i> gene (93.0% sequence similarity across our dataset) alone could be used as a fast and practical means for identifying species within <i>Hyphomonas</i>. Finally, <i>Hyphomonas</i>' geographic distribution shows that strains from the same area tend to cluster together as discrete species. This study provides a framework for the discrimination and phylogenetic analysis of the genus <i>Hyphomonas</i> for the first time, and will contribute to a more thorough understanding of the biological and ecological roles of this genus.</p></div

Phylogenetic tree based on concatenated housekeeping genes.

<p>Percentage bootstrap values over 50% (1000 replicates) are indicated on internal branches. Blank circles show nodes that were also recovered in maximum-likelihood and maximum-parsimony trees based on the same sequences. Bar, 0.05 nucleotide substitution rate (Knuc) units. <i>Hirschia beltic</i>a ATCC 49814^T (NC_012982) was used as the outgroup. Water depth is represented by color (0–1000 m, blue color; >1000 m, black color; unknown depth, red color.). No symbol: no detailed information about the source. Bold font strain names indicate their genomes are available.</p

Bacterial strains used in this study.

<p>ND, no data; MCCC, Marine Culture Collection of China;</p><p>a, refer to Leifson, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101394#pone.0101394-Leifson1" target="_blank">[61]</a>;</p><p>b, refer to Weiner et al, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101394#pone.0101394-Weiner3" target="_blank">[62]</a>.</p

Intraspecies and interspecies similarity ranges of 16S rDNA and housekeeping genes in <i>Hyphomonas</i>.

<p>Intraspecies and interspecies similarity ranges of 16S rDNA and housekeeping genes in <i>Hyphomonas</i>.</p

Neighbour-joining tree showing the phylogeny of 42 <i>Hyphomonas</i> strains, based on the 16S rRNA gene sequences.

<p>Percentage bootstrap values over 50% (1000 replicates) are indicated on internal branches. Filled circles show nodes that were also recovered in maximum-likelihood and maximum-parsimony trees based on the same sequences. Bar, 0.01 nucleotide substitution rate (Knuc) units. <i>Hirschia beltica</i> ATCC 49814^T (NR_074121) was used as the outgroup.</p