Ethics statement

No specific permissions were required for collection of these the bacterial strains used in phylogenetic analysis in this study, as they are isolated from areas beyond national jurisdiction or from areas within the exclusive economic zone of China. Moreover, the sample sampling did not involve endangered or protected species.

Bacterial strains

A total of 76 strains of 5 species close to B. pumilus were chosen for the phylogeny study: 15 from the Pacific Ocean, 7 from the Indian Ocean, 3 from the Atlantic Ocean, 7 from the North Polar Region, 20 from the coast area of Fujian Province, 4 from the East China Sea and the Yellow Sea and 20 from the South China Sea (Table 1 and Figure S1 in File S1). These strains were deposited at Marine Culture Collection of China (MCCC).

In addition, 3 type strains, B. pumilus DSM 27^T isolated from soil [19], B. safensis FO-36b^T isolated by the Jet Propulsion Laboratory spacecraft-assembly facility of California in USA [20] and B. altitudinis 41KF2b^T isolated from air samples of high elevations (41,000 m) in India [21], were also included in the phylogeny study; these strains were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) in Germany. Unfortunately, 2 other type strains, B. stratosphericus and B. aerophilus, isolated from the same sample as B. altitudinis 41KF2b^T, are no longer available in public collections or from the authors and therefore not included in the our analyses. The gyrB sequences of 73 strains were acquired from the NCBI database, and their detailed information is listed in Table S1 in File S1.

DNA extraction

The strains were reactivated on a modified solid Luria-Bertani medium (10 g peptone, 5 g yeast extract, 10 g NaCl, 15 g agar and 1 L double-distilled water, pH 7.5) [22] and incubated at 37°C for 24 h. A suitable amount of cells on the plates were selected and transferred to 1.5 mL centrifuge tubes using sterile pipette tips. Genomic DNA was extracted using the SBS extraction kit (SBS Genetech Co., Ltd. in Shanghai, China) according to the manufacturer's instructions.

PCR amplification and sequencing of 16S rRNA and housekeeping genes

The 16S rRNA gene was amplified by PCR using universal primers 27F and 1492R, and seven housekeeping genes were amplified using specific primers designed using Primer 5.0 (Table S2 in File S1). The genes were amplified under nearly the same conditions. In brief, each PCR mixture contained 1 µL genomic DNA, 1.25 U Ex Taq^TM DNA polymerase (TaKaRa), 4 µL dNTP mixture (2.5 mM of each dNTP), 1 µL each primer (20 µM), 5 µL 10×Ex Taq buffer (Mg²⁺ Plus) and sterile deionized water to a total volume of 50 µL. PCR was performed using a My Gen^TM L Series Peltier Thermal Cycle (Hangzhou Long Gene Scientific Instruments Co., Ltd, China). Each PCR product was separated by electrophoresis on a 1% agarose gel. The target PCR products were purified with the AxyPrep^TM PCR Clean up kit (Axygen Scientific, Inc., USA) according to the manufacturer's instructions and sequenced using the ABI3730xl platform (BGI Co., Ltd, China).

The assembly and modification of the DNA sequences, including the 16S rRNA gene and seven housekeeping genes, were performed using DNAMAN 5.0 software. All sequences were deposited into the GenBank database; the accession numbers were listed in Table S3 in File S1.

Phylogenetic analysis based on single gene analysis and MLSA

The determined sequences of the 16S rRNA gene and seven housekeeping genes were analyzed against sequences in the NCBI database using Blastn [23]. A substitution saturation assessment was performed for each gene sequence using DAMBE [24]. Recombination events in the DNA sequence alignments were evaluated using RDP 3.0 [25]. The genetic distances and sequence similarities of gene(s) were calculated using Kimura’s 2-parameter model [26] with the MEGA 5.0 software. The selective pressure on housekeeping gene was evaluated with the calculation of nonsynonymous (Ka) and synonymous (Ks) substitution rates （Ka/Ks）by the DnaSP 5.0 software [27].

The phylogenetic trees were constructed using the neighbor-joining (NJ) algorithm [28] with MEGA 5.0 [29]. The strengths of the internal branches of the resulting trees were statistically evaluated by bootstrap analysis with 1000 bootstrap replications. B. cereus ATCC 14579^T (GenBank accession: AE016877) was used as the outgroup.

Phylogenetic diversity revealed by 16S rRNA gene analysis

All the tested bacteria were subjected to a 16S rRNA gene analysis, even though they had been characterized (approximately 600 bp) prior to deposition in MCCC. Nearly the full-length 16S rRNA gene sequences (approximately 1513 bp) were obtained to further assess the taxonomic affiliation and phylogeny of the strains.

The results demonstrated that the genetic distance of the 16S rRNA gene ranged from 0-0.005 (mean 0.002). Moreover, the number of alleles and polymorphic sites were only 7 and 10, respectively, and the proportion of polymorphic sites was 0.7%. In addition, the intraspecies similarities of 16S rRNA gene ranged from 99.6% to 100%, while the interspecies similarities were 99.5%-100%. These features of the 16S rRNA gene were presented in Table 2 and Table S4 in File S1. The 16S rRNA genes of the strains were highly conserved, and their similarities had overlap in intraspecies and interspecies, therefore it was unsuitable for the differentiation of these closely related strains.

Despite the high similarity, the phylogenetic tree of the 16S rRNA gene showed that the 79 strains were divided into two groups (Figure 1). The large group contained our 52 isolates, which were close to the type strain B. altitudinis; the small group contained 24 strains that we isolated, which were close to the type strains B. pumilus and B. safensis and cannot be distinguished by their 16S rRNA gene.

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Figure 1. Phylogenetic tree based on the 16S rRNA genes of marine bacteria belonging to the B. pumilus group.

The tree was constructed using the neighbor-joining method with MEGA 5.0. Bootstrap values over 50% (1000 replications) were shown at each node. Bar, % estimated substitution. B. cereus ATCC 14579^T was used as the outgroup.

https://doi.org/10.1371/journal.pone.0080097.g001

Characteristics of seven housekeeping genes

To discriminate among the closely related bacteria, the housekeeping genes gyrB, rpoB, aroE, mutL, pycA, pyrE and trpB were chosen for analysis; 79 strains, including the 3 type strains, were analyzed. The characteristics of each housekeeping gene, such as the gene length, number of alleles, polymorphic sites, the mean G+C content, the genetic distance and the similarity range were shown in Table 2 and Table S4 in File S1.

The correlation of genetic distance between two housekeeping genes was calculated (Table S5 in File S1). An analysis of the characteristics and genetic distance of the housekeeping genes (Table 2) demonstrated that all the housekeeping genes showed remarkably higher resolution than the 16S rRNA gene (Table 2). Among the 7 housekeeping genes, the pyrE gene exhibited the highest resolution, with 29.3% polymorphic sites and the largest genetic distance range (0-0.179), whereas rpoB exhibited the lowest resolution. Although mutL had a higher allele number (38) than other genes, its polymorphic site percentage was less than many others. Specifically, gyrB displayed a better differentiation among strains close to B. altitudinis; in contrast, aroE was more powerful for strains of B. pumilus, and pyrE was better for strains of B. safensis (Table S6 in File S1).

Further, DNA sequence similarity ranges of the 7 genes at intraspecies and interspecies levels were analyzed with the MEGA 5.0 software. These 79 strains were divided into 6 species, three of which were established species and others were potential novel species as documented below. The similarity ranges at intraspecies and interspecies levels were shown in Table S4 in File S1. An obvious gap between intraspecies and interspecies similarity ranges was observed in most housekeeping genes with exceptions of 16S rDNA and rpoB (Figure 2, Table S4 in File S1). For more details, the numbers of strain pairs within different similarity grades of the housekeeping genes of the 79 strains were shown in Table S7 and Figure S2 in File S1. These data indicates that 16S rDNA and rpoB were inappropriate for species discrimination among the bacteria of this group, while other genes showed a general interspecies similarity gap of 92% to 96%, and can serve in species discrimination, especially pyrE (92%-95%) and aroE (93-95%) Table S7 and Figure S2 in File S1),

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Figure 2. Intraspecies and interspecies similarity ranges of housekeeping genes in the B. pumilus group.

https://doi.org/10.1371/journal.pone.0080097.g002

In addition, the Ka/Ks ratio of each housekeeping gene of different species and all the 79 strains was calculated, the results were displayed in Table S8 and Figure S3 in File S1. All the genes exhibited low Ka/Ks ratios ranging from 0.0000-0.1200 (Table S8 and Figure S3 in File S1), suggesting that they are under negative selection pressure. However, the ratios of Ka/Ks of each gene in different species were significant differences. The pyrE gene had the highest Ka/Ks ratio (0.1200) in B. altitudinis, while the gene aroE in B. pumilus and B. safensis were the highest, respectively 0.0800 and 0.0561. Even at interspecies level based on all the 79 strains, pyrE had the highest Ka/Ks ratio (0.0731). In contrast, rpoB had the lowest the Ka/Ks ratio, and was the most conserved among the seven housekeeping genes.

Phylogenetic diversity revealed by individual housekeeping genes

Prior to the phylogenetic analysis, these housekeeping genes were subjected to an examination of sequence substitution saturation and recombination events (data not shown). The saturation test of each housekeeping gene with DAMBE showed no sign of substitution saturation, and no recombination events were found in any of their tested housekeeping genes, as determined by program RDP-3.0. These results indicated that these sequences provided essential phylogenetic information.

Phylogenetic analyses based on each of the housekeeping genes were able to distinguish the strains at the species level. Moreover, the phylogenetic trees possessed nearly congruent topology structure (Figure S4-S10 in File S1). Specifically, the 79 strains were divided into 6 groups from A to F. Group A is the largest, containing 49 strains close to B. altitudinis; Group F is the second largest group, containing 13 strains belonging to B. safensis. Group D consisted of 13 strains attributed to B. pumilus. Additional three minor groups were revealed, Groups B, C and E, supported by only 1 to 2 strains each. These minorities represent putative novel taxa.

Slight differences were also observed in some groups among the topologies of the seven trees. For example, B. pumilus was close to B. altitudinis in the phylogenetic tree of gyrB; in contrast, B. pumilus is close to B. safensis in other trees. In addition, the position of Groups B, C and D varied in the trees of gyrB, rpoB and mutL. For instance, Group B was closer to Group A in the phylogenetic trees of gyrB, aroE, mutL, pyrE and trpB, whereas Group B was closer to the groups in the large cluster of B. safensis and B. pumilus in the tree of pycA. Other small differences were also observed in the trees, as shown in the supplementary materials (Figure S4-S10 in File S1).

Phylogeny based on the concatenated housekeeping genes

The seven housekeeping genes were concatenated in the order of gyrB-rpoB-pycA-pyrE-mutL-aroE-trpB (5649 bp) to reexamine the phylogeny of the 79 strains (Figure 3). The new phylogenetic tree showed a similar topology as the trees described above based on a single gene but was more elaborate and stable.

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Figure 3. Phylogenetic tree based on seven housekeeping genes concatenated of marine isolates of the B. pumilus group.

The tree was constructed using the neighbor-joining method with MEGA 5.0. Bootstrap values over 50% (1000 replications) were shown at each node. Bar, % estimated substitution. B. cereus ATCC 14579^T was used as the outgroup.

https://doi.org/10.1371/journal.pone.0080097.g003

Specifically, Group A consisted of 49 strains belonging to B. altitudinis that could be divided into 37 genetic types from A1 to A37 (Table 1). Group D contained 13 strains belonging to B. pumilus, with 7 genetic types, D1 to D7 (Table 1); Group F also contained 13 strains of 9 genetic types, F1 to F9 (Table 1), and belonging to B. safensis. In contrast, fewer bacteria were allotted into Group B, Group C and Group E and could not be assigned to any the described species due to low similarity. For example, the only strain in Group B showed 92.12%, 89.22% and 89.50% similarity with B. altitudinis, B. pumilus and B. safensis and a genetic distance of 0.079, 0.108 and 0.105, respectively. Both strains in Group C shared 91.04%, 90.21% and 91.02% similarity with the above type strains, with a genetic distance 0.09, 0.098 and 0.09, respectively. Similarly, the only member of Group E shared 89.48%, 91.41% and 93.93% similarity with the three type strains and a genetic distance of 0.105, 0.086 and 0.061, respectively. These unassigned strains represent novel bacterial taxa.

Correlation between phylogenetic and geographic distribution

The geographical distribution of the 76 strains covered various marine environments: a subtropical coastal area, the Pacific Ocean, the Indian Ocean, the Arctic Ocean, the Atlantic and the South China Sea.

Among these bacteria, those belonging to B. altitudinis were in the majority and had the widest geographical distribution; 48 of our isolates were allocated to this group in the concatenated gene tree (Group A in Figure 3). These isolates were mainly from three areas, the coastal area (○), South China Sea (□) and Pacific ocean (△), though some were isolated from the Indian Ocean (▽), the Atlantic Ocean (☆) and the Arctic Ocean. (◇). Group D contained twelve strains that were isolated from a Fujian coastal area and pelagic areas; however, no strain originated from the Arctic Ocean (◇) or South China Sea (□). The 12 strains in Group F were mainly from the coast area (○),South China Sea (□) and Arctic Ocean (◇). Among the above-mentioned special clades composed of putative novel species, two of three are from marine aquiculture environments, one from fish gut (strain 1 in Group B) and another from a shrimp farm (strain 70 in Group C).

In addition, according to the water depth, the habitats were arbitrarily divided into the upper layer (0-1000 m) and deep layer (>1000 m) and marked in green and black, respectively, in the phylogenetic tree (Figure 3). According to the tree, it was observed that the strains tended to cluster together to some extent according to the water depth. For example, in the largest group (Group A, B. altitudinis), bacteria from shallow areas tended to cluster together (in green). On the other hand, bacteria from the deep sea (in black) tended to cluster. Further, a principal component analysis (PCA) based on all strains was carried out to examine the key factors influencing their distribution using unweighted UniFrac. However, the correlation of the phylogenetic and geographic distribution was not significant (data not shown). This may be due to the inadequate strain numbers in other species.

To compare with their terrestrial counterparts, more sequences of the gyrB gene of the B. pumilus group were retrieved from GenBank (much less data for other housekeeping genes are available), and a phylogenetic tree of 152 strains was constructed (Figure 4). In general, the topological structure of the tree was the same as that constructed with our bacteria alone (Figure 3), though the three clades containing the potential novel species remain as minorities in the new tree. Some mistakes in nomenclature were observed for some strains retrieved from NCBI, such as strains 99, 101, 107, 108, 113 and 116, which actually belong to B. altitudinis rather than B. pumilus, as described in NCBI.

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Figure 4. Phylogenetic tree based on gyrB genes of 152 strains of both marine and terrestrial origins.

The tree was constructed using the neighbor-joining method with MEGA 5.0. Bootstrap values over 50% (1000 replications) were shown at each node. Bar, % estimated substitution. B. cereus ATCC 14579^T was used as the outgroup. The number represented the number of strains in each portion of the pie chart. The bacteria from marine environments were in blue, and the others were in red. The pie charts illustrated the proportions of marine and terrestrial origins in each large cluster.

https://doi.org/10.1371/journal.pone.0080097.g004

Of note, based on the large tree of gyrB genes, the bacteria of marine origin tended to cluster together; with some exceptions, the strains of terrestrial origin also clustered together (Figure 4). Most of our marine isolates were placed in the large cluster of B. altitudinis, positioned as a separate clade (numbers in blue); a similar tendency was observed for the B. pumilus and B. safensis clusters. Most of the terrestrial bacteria were allotted to B. safensis, forming a distinct clade (in red).