Vibrio plantisponsor sp. nov., a diazotrophic bacterium isolated from a mangrove associated wild rice (Porteresia coarctata Tateoka)

Two Gram negative, facultatively anaerobic, halophilic, motile, slightly curved rod-shaped bacterial strains MSSRF60^T and MSSRF64 were isolated from the roots of a mangrove-associated wild rice collected in the Pichavaram mangroves, India. These strains possess the key functional nitrogenase gene nifH. Phylogenetic analysis based on the 16S rRNA, recA, gapA, mreB, gyrB and pyrH, gene sequences revealed that strains MSSRF60^T and MSSRF64 belong to the genus Vibrio, and had the highest sequence similarity with the type strains of Vibrio diazotrophicus LMG 7893^T (99.7, 94.8, 98.5, 97.9, 94.0 and 90.7%, respectively), Vibrio areninigrae J74^T (98.2, 87.5, 91.5, 88.9, 86.5 and 84.6% respectively) and Vibrio hispanicus LMG 13240^T (97.8, 87.1, 91.7, 89.8, 84.1 and 81.9%, respectively). The fatty acid composition too confirmed the affiliation of strains MSSRF60^T and MSSRF64 to the genus Vibrio. These strains can be differentiated from the most closely related Vibrio species by several phenotypic traits. The DNA G + C content of strain MSSRF60^T was 41.8 mol%. Based on phenotypic, chemotaxonomic, genotypic (multilocus sequence analysis using five genes and genomic fingerprinting using BOX-PCR) and DNA–DNA hybridization analyses, strains MSSRF60^T and MSSRF64 represent a novel species of the genus Vibrio, for which the name Vibrio plantipsonsor sp. nov. is proposed. The type strain is MSSRF60^T (=DSM 21026^T = LMG 24470^T = CAIM 1392^T).     

Vibrio celticus sp. nov., a new Vibrio species belonging to the Splendidus clade with pathogenic potential for clams

A group of four motile facultative anaerobic marine isolates (Rd 8.15^T [=CECT 7224^T, =LMG 23850^T], Rd 16.13, Rd 6.8 [=LMG 25696] and Rd2L5) were obtained from cultured clams (Ruditapes philippinarum and Venerupis pullastra) in Galicia, north-western Spain. They formed a tight phylogenetic group based on sequences of the 16S rRNA gene and the four housekeeping genes rpoA (encoding the α-chain of RNA polymerase), rpoD (encoding the sigma factor of RNA polymerase), recA (encoding RecA protein), and atpA (encoding the α-subunit of bacterial ATP synthase). The phylogenies based on these sequences indicated that the four isolates represented a novel species in the genus Vibrio, and more precisely in the Splendidus clade. DNA–DNA hybridizations with the type strains of species showing more than 98.6% 16S rRNA gene sequence similarity, revealed a DNA–DNA relatedness below 70%. The isolates could be differentiated from the phylogenetically related Vibrio species on the basis of several phenotypic features. In addition, strain Rd 8.15^T showed potential pathogenic activity for adult clams in virulence assays. The name Vibrio celticus sp. nov. is proposed for this new taxon, with the type strain being Rd 8.15^T (=CECT 7224^T, =LMG 23850^T).     

Photobacterium piscicola sp. nov., isolated from marine fish and spoiled packed cod

Five isolates from marine fish (W3^T, WM, W1S, S2 and S3) and three isolates misclassified as Photobacterium phosphoreum, originating from spoiled modified atmosphere packed stored cod (NCIMB 13482 and NCIMB 13483) and the intestine of skate (NCIMB 192), were subjected to a polyphasic taxonomic study. Phylogenetic analysis of 16S rRNA gene sequences showed that the isolates were members of the genus Photobacterium. Sequence analysis using the gapA, gyrB, pyrH, recA and rpoA loci showed that these isolates formed a distinct branch in the genus Photobacterium, and were most closely related to Photobacterium aquimaris, Photobacterium kishitanii, Photobacterium phosphoreum and Photobacterium iliopiscarium. The luxA gene was present in isolates W3^T, WM, W1S, S2 and S3 but not in NCIMB 13482, NCIMB 13483 and NCIMB 192. AFLP and (GTG)₅-PCR fingerprinting indicated that the eight isolates represented at least five distinct genotypes. DNA–DNA hybridizations revealed 89% relatedness between isolate W3^T and NCIMB 192, and values below 70% with the type strains of the phylogenetically closest species, P. iliopiscarium LMG 19543^T, P. kishitanii LMG 23890^T, P. aquimaris LMG 26951^T and P. phosphoreum LMG4233^T. The strains of this new taxon could also be distinguished from the latter species by phenotypic characteristics. Therefore, we propose to classify this new taxon as Photobacterium piscicola sp. nov., with W3^T (=NCCB 100098^T = LMG 27681^T) as the type strain.     

Kiloniella majae sp. nov., isolated from spider crab (Maja brachydactyla) and pullet carpet shell clam (Venerupis pullastra)

Ten Gram-negative, rod-shaped and motile bacterial strains were isolated from spider crab (M27.10, M27.11a, F36.1, F36.4, M56.1, F76.17b, M146.1, M166.3 and M166.6) and pullet carpet shell clam (SBRF 1.10) collected in the coast of Galicia. Analyses of the 16S rRNA genes showed that the strains belong to the genus Kiloniella and have high similarity with the species Kiloniella spongiae (99.44–99.86%) and Kiloniella litopenaei (99.0–99.5%). Strains M56.1^T (=CECT 9195, =LMG 29925), M146.1 (=CECT 9193, =LMG 29926) and SBRF 1.10 (=CECT 9194, =LMG 29927) were selected on the basis of genotyping by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Phylogenetic analysis based on concatenated sequences of the genes gyrB, ftsZ, rpoD and mreB showed that the isolates form a differentiated branch within the genus Kiloniella. Moreover, the average nucleotide identity (ANIm, ANIb and OrthoANI) and in silico estimated DNA–DNA reassociation values between selected Galician isolates and Kiloniella species were below the established cut-off for species deliniation. The results obtained in the genetic and phenotypical analyses indicate that the isolates represent a new species of the genus Kiloniella, for which the name Kiloniella majae sp. nov. is proposed with strain M56.1^T (=CECT 9195^T, =LMG 29925^T) as the type strain.     

Vibrio taketomensis sp. nov. by genome taxonomy

Two novel strains C4III282^T and C4III291 were isolated from seawater collected a site off the Taketomi coral reef. Phylogenetic analysis based on the 16S rRNA sequences revealed that the two strains belong to the genus Vibrio. MLSA using eight protein-coding genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) showed that C4III282^T and C4III291 are closely related to the members of the Ponticus clade, namely Vibrio panuliri JCM 19500^T, Vibrio ponticus DSM 16217^T, and “Vibrio rhodolitus” G98. ANI and in silico DDH values with members of the Ponticus clade were 77.6-78.7% and 22.2-23.1, respectively. The name Vibrio taketomensis sp. nov. is proposed with C4III282^T (CAIM 1928^T = DSM 106943^T = JCM 33434^T) as the type strain.     

Multilocus sequence analysis of putative Vibrio mediterranei strains and description of Vibrio thalassae sp. nov.

A multilocus sequence analysis based on partial gyrB, mreB, rpoD and pyrH genes was undertaken with 61 putative Vibrio mediterranei/V. shilonii strains from different hosts (mussels, oysters, clams, coral, fish and plankton) or habitat (seawater and sediment) and geographical origins (Mediterranean, Atlantic and Pacific). A consistent grouping was obtained with individual and concatenated gene sequences, and the clade, comprising 54 strains, was split into three subclades by all methods: subclade A (40 strains, including AK1, the former type strain of Vibrio shilonii), subclade B (8 strains) corresponding to the species V. mediterranei, and subclade C (six strains) representing a new species, V. thalassae sp. nov., with strain MD16^T (=CECT 8203^T = KCTC 32373^T) as the proposed type strain.     

Arcobacter cloacae sp. nov. and Arcobacter suis sp. nov., two new species isolated from food and sewage

Three strains recovered from mussels (F26), sewage (SW28-13^T) and pork meat (F41^T) were characterized as Arcobacter. They did not appear to resemble any known species on the basis of their 16S rDNA-RFLP patterns and the rpoB gene analyses. However, strains F26 and SW28-13^T appeared to be the same species. The 16S rRNA gene sequence similarity of strains SW28-13^T and F41^T to the type strains of all other Arcobacter species ranged from 94.1% to 99.6% and 93.4% to 98.8%, respectively. Phenotypic characteristics and the DNA–DNA hybridization (DDH) results showed that they belonged to 2 new Arcobacter species. A multilocus phylogenetic analysis (MLPA) with the concatenated sequences of 5 housekeeping genes (gyrA, atpA, rpoB, gyrB and hsp60) was used for the first time in the genus, showing concordance with the 16S rRNA gene phylogenetic analysis and DDH results. The MALDI-TOF mass spectra also discriminated these strains as two new species. The names proposed for them are Arcobacter cloacae with the type strain SW28-13^T (=CECT 7834^T = LMG 26153^T) and Arcobacter suis with the type strain F41^T (=CECT 7833^T = LMG 26152^T).     

Paraburkholderia gardini sp. nov. and Paraburkholderia saeva sp. nov.: Novel aromatic compound degrading bacteria isolated from garden and forest soil samples

Three forest and four botanical garden top soil isolates with unique MALDI-TOF mass spectra were identified as Paraburkholderia strains closely related to Paraburkholderia sartisoli through recA gene sequence analysis. OrthoANIu, digital DNA-DNA hybridization analyses and phylogenomic analyses demonstrated that the five strains represented two new Paraburkholderia species closely related to P. sartisoli. The genome of strain LMG 31841^T had a cumulative size of 6.3 Mb and a G + C content of 62.64 mol%; strain LMG 32171^T had a genome size of 5.8 Mb and a G + C content of 62.91 mol%. Hemolysis on horse blood agar, beta-galactosidase and phosphoamidase activity, and assimilation of adipic acid and trisodium citrate allowed phenotypic differentiation of strains LMG 31841^T, LMG 32171^T and P. sartisoli LMG 24000^T. An analysis of the genomic potential for aromatic compound degradation yielded additional differences among strains representing these three species, but also highlighted some discrepancies between the presence of genes and pathways, and the phenotype revealed through growth experiments using a mineral salts medium supplemented with single aromatic compounds as carbon sources. We propose to classify all isolates from the present study into two novel Paraburkholderia species, for which we propose the names Paraburkholderia gardini with LMG 32171^T (=CECT 30344^T) as the type strain, and Paraburkholderia saeva with LMG 31841^T (=CECT 30338^T) as the type strain.     

Chromohalobacter moromii sp. nov., a moderately halophilic bacterium isolated from lupine-based moromi fermentation

Three moderately halophilic strains, TMW 2.2308^T, TMW 2.2299 and TMW 2.2304, were isolated from a lupine-based moromi fermentation. Initial identification based on their low molecular sub-proteome using mass spectrometry showed relation to the genus Halomonas, however, low score values indicated novelty. The comparison of 16S rRNA gene sequences placed these strains within the genus Chromohalobacter with C. japonicus CECT 7219^T (99.67% 16S rRNA sequence similarity to strain TMW 2.2308^T), C. canadensis DSM 6769^T (99.54%) and C. beijerinckii LMG 2148^T (99.32%) being their closest relatives. However, average nucleotide highest identity values of TMW 2.2308^T to C. beijerinckii LMG 2148^T of 93.12% and 92.88% to C. japonicus CECT 7219^T demonstrate that it represents a novel species within the genus Chromohalobacter with additional strains TMW 2.2299 (96.91%) and TMW 2.2304 (96.98%). The isolated strains were non-spore-forming, motile and able to grow at temperatures from 5 to 45 °C with an optimum at 37 °C. Growth of TMW 2.2308^T occurs at 5 to 25% (w/v) NaCl with optimum growth between 10 and 12.5%. The genome of TMW 2.2308^T has a size of 3.47 Mb and a G + C content of 61.0 mol%. The polyphasic evidence lead to the classification of TMW 2.2308^T, TMW 2.2299 and TMW 2.2304 as members of a novel species of the genus Chromohalobacter. We propose a novel species as Chromohalobacter moromii sp. nov., with TMW 2.2308^T (=DSM 113153^T =CECT 30422^T) as the type strain.     

Vibrio gallaecicus sp. nov. isolated from cultured clams in north-western Spain

A group of three motile facultative anaerobic marine bacteria were isolated from cultured Manila clams (Ruditapes philippinarum) in Galicia, north-western Spain. The strains were characterized phenotypically and genotypically. Phylogenetic analysis of the 16S rRNA gene and four housekeeping genes, RNA polymerase α-chain (rpoA), RecA protein (recA), the α-subunit of bacterial ATP synthase (atpA) and the uridine monophosphate (UMP) kinase (pyrH), indicated that these strains were closely related to the Vibrio splendidus clade. The amplified fragment length polymorphism (AFLP) fingerprints, DNA–DNA hybridizations and phylogenies of the housekeeping and 16S rRNA gene sequences showed that the three strains represented a different species from all currently described vibrios. The new species could be differentiated from its nearest neighbours on the basis of several phenotypic features. The three strains are therefore a novel species within the genus Vibrio, for which the name Vibrio gallaecicus is proposed, with the type strain being VB 8.9^T(=CECT 7244^T=LMG 24045^T).     

Photobacterium malacitanum sp. nov., and Photobacterium andalusiense sp. nov., two new bacteria isolated from diseased farmed fish in Southern Spain

Three strains, H01100409B^T, H01100413B, and H27100402H^T, were isolated from several internal organs of diseased redbanded seabream (Pagrus auriga) reared in Andalusia (Southern Spain). All strains were studied by phenotypic, including chemotaxonomy, and genomic characteristics. Phylogenetic analysis based on concatenated sequences of six housekeeping genes (gyrB, ftsZ, topA, mreB, gapA, and 16S rRNA) supported the inclusion of the strains within the clade Phosphoreum of the genus Photobacterium, and two of the strains (H27100402H^T and H01100409B^T) formed a tight group separated from the closest species P. aquimaris. Genomic analyses, including average nucleotide identity (ANIb and ANIm) and DNA–DNA hybridization (DDH), clearly separated strains H27100402H^T and H01100409B^T from the other species within the clade Phosphoreum with values below the thresholds for species delineation. The chemotaxonomic features (including FAME analysis and MALDI-TOF-MS) of H27100402H^T and H01100409B^T strains confirmed their differentiation from the related taxa. The results demonstrated that strain H01100413B was classified as P. aquimaris and the strains H27100402H^T and H01100409B^T represented a new species each in the genus Photobacterium, for which we propose the names Photobacterium malacitanum sp. nov., type strain H27100402H^T (=CECT 9190^T = LMG 29992^T), and Photobacterium andalusiense sp. nov., type strain H01100409B^T (=CECT 9192^T = LMG 29994^T).     

Description of the two novel species of the genus Helicobacter: Helicobacter anatolicus sp. nov., and Helicobacter kayseriensis sp. nov., isolated from feces of urban wild birds

A total of 26 Gram-negative, motile, gently curved, and rod-shaped isolates were recovered, during a study to determine the faeco-prevalence of Helicobacter spp. in urban wild birds. Pairwise comparisons of the 16S rRNA gene sequences indicated that these isolates belonged to the genus Helicobacter and phylogenetic analysis based on the 16S rRNA gene sequences showed that the isolates were separated into two divergent groups. The first group consisted of 20 urease-positive isolates sharing the highest 16S rRNA gene sequence identity levels of 98.5–98.6% to H. mustelae ATCC 43772^T, while the second group contained six urease-negative isolates with the sequence identity level of 98.5% to the type strain of H. pametensis ATCC 51478^T. Five isolates were chosen and subjected to comparative whole-genome analysis. The phylogenetic analysis of the 16S rRNA, gyrA and atpA gene sequences showed that Helicobacter isolates formed two separate phylogenetic clades, differentiating the isolates from the other Helicobacter species. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses between strains faydin-H8^T, faydin-H23^T and their close neighbors H. anseris MIT 04-9362^T and H. pametensis ATCC 51478^T, respectively, confirmed that both strains represent novel species in the genus Helicobacter. The DNA G+C contents of the strains faydin-H8^T and faydin-H23^T are 32.0% and 37.6%, respectively. The results obtained for the characterization of the wild bird isolates indicate that they represent two novel species, for which the names Helicobacter anatolicus sp. nov., and Helicobacter kayseriensis sp. nov., are proposed, with faydin-H8^T(=LMG 32237^T = DSM 112312^T) and faydin-H23^T(=LMG 32236^T = CECT 30508^T) as respective type strains.     

Phylogeny and Molecular Identification of Vibrios on the Basis of Multilocus Sequence Analysis

We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.     

Aliarcobacter vitoriensis sp. nov., isolated from carrot and urban wastewater

Two isolates, one recovered from a carrot and another one from urban wastewater, were characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both isolates clustered together, and were most closely related to Aliarcobacter lanthieri. Multilocus phylogenetic analysis (MLPA) using the concatenated sequences of five housekeeping genes (atpA, gyrA, gyrB, hsp60 and rpoB) suggested that these isolates formed a distinct phylogenetic lineage among the genera derived from the former genus Arcobacter. Whole-genome sequence, in silico DNA-DNA hybridization (isDDH) and the average nucleotide identity (ANI) value between the genome of strain F199^T and those of related species confirmed that these isolates represent a novel species. These strains can be differentiated from its phylogenetically closest species A. lanthieri by its inability to growth on 1% glycine and by their enzyme activity of esterase lipase (C8) and acid phosphatase. Our results, by the application of a polyphasic analysis, confirmed that these two isolates represent a novel species of the genus Aliarcobacter, for which the name Aliarcobacter vitoriensis sp. nov. is proposed. The type strain is F199^T (=CECT 9230^T=LMG 30050^T).     

Vibrio zhuhaiensis sp. nov., isolated from a Japanese prawn (Marsupenaeus japonicus)

A Gram-negative, oxidase-positive, facultatively anaerobic bacterium, designated strain E20121, was isolated from the digestive tract of a Japanese prawn (Marsupenaeus japonicus) collected from the coastal sea water area of Zhuhai, Guangdong province, China. The new isolate was determined to be closely related to Vibrio ponticus DSM 16217^T, having 97.6 % 16S rRNA gene sequence similarity. Phylogenetic analysis based on recA, pyrH and rpoA also showed low levels of sequence similarities (72.6–96.6 %) with all species of the genus Vibrio. A multigene phylogenetic tree using concatenated sequences of the four genes (16S rRNA, rpoA, recA and pyrH) clearly showed that the new isolate is different from the currently known Vibrio species. DNA–DNA hybridization experiments revealed similarity values below 70 % with the closest related species V. ponticus DSM 16217^T. Several phenotypic traits enabled the differentiation of strain E20121 from the closest phylogenetic neighbours. The DNA G+C content of strain E20121 was determined to be 47.6 mol % and the major fatty acid components identified were C_16:1ω7c and/or C_16:1ω6c (39.8 %), C_18:1ω7c (13.6 %) and C_16:0 (9.6 %). Based on genotypic, phenotypic, chemotaxonomic, phylogenetic and DNA–DNA hybridization analyses, strain E20121 is proposed to represent a novel species of the genus Vibrio for which the name Vibrio zhuhaiensis sp. nov. is proposed. The type strain is E20121^T(=DSM 25602^T = CCTCC AB 2011174^T).     

Gilliamella intestini sp. nov., Gilliamella bombicola sp. nov., Gilliamella bombi sp. nov. and Gilliamella mensalis sp. nov.: Four novel Gilliamella species isolated from the bumblebee gut

Spectra of five isolates (LMG 28358^T, LMG 29879^T, LMG 29880^T, LMG 28359^T and R-53705) obtained from gut samples of wild bumblebees of Bombus pascuorum, Bombus lapidarius and Bombus terrestris were grouped into four MALDI-TOF MS clusters. RAPD analysis revealed an identical DNA fingerprint for LMG 28359^T and R-53705 which also grouped in the same MALDI-TOF MS cluster, while different DNA fingerprints were obtained for the other isolates.Comparative 16S rRNA gene sequence analysis of the four different strains identified Gilliamella apicola NCIMB 14804^T as nearest neighbour species. Average nucleotide identity values of draft genome sequences of the four isolates and of G. apicola NCIMB 14804^T were below the 96% threshold value for species delineation and all four strains and G. apicola NCIMB 14804^T were phenotypically distinct. Together, the draft genome sequences and phylogenetic and phenotypic data indicate that the four strains represent four novel Gilliamella species for which we propose the names Gilliamella intestini sp. nov., with LMG 28358^T as the type strain, Gilliamella bombicola sp. nov., with LMG 28359^T as the type strain, Gilliamella bombi sp. nov., with LMG 29879^T as the type strain and Gilliamella mensalis sp. nov., with LMG 29880^T as the type strain.     

Gluconacetobacter maltaceti sp. nov., a novel vinegar producing acetic acid bacterium

Comparison of HaeIII- and HpaII-restriction profiles of PCR-amplified 16S-23S rDNA ITS regions of Gluconacetobacter sp. LMG 1529^T and SKU 1109 with restriction profiles of reference strains of acetic acid bacteria described by Tr?ek and Teuber [34] revealed the same but unique restriction profiles for LMG 1529^T and SKU 1109. Further analyses of nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rDNA ITS sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated both strains to a single phylogenetic cluster well separated from the other species of the genus Gluconacetobacter. DNA–DNA hybridizations confirmed their novel species identity by 73% DNA–DNA relatedness between both strains, and values below the species level (<70%) between SKU 1109 and the type strains of the closest phylogenetic neighbors. The classification of strains LMG 1529^T and SKU 1109 into a single novel species was confirmed also by AFLP and (GTG)₅-PCR DNA fingerprinting data, as well as by phenotypic data. Strains LMG 1529^T and SKU 1109 can be differentiated from their closely related Gluconacetobacter species, Gluconacetobacter entanii and Gluconacetobacter hansenii, by their ability to form 2-keto-d-gluconic acid from d-glucose, their ability to use d-mannitol, d-gluconate and glycerol as carbon source and form acid from d-fructose, and their ability to grow without acetic acid. The major fatty acid of LMG 1529^T and SKU 1109 is C_18:1ω7c (60.2–64.8%). The DNA G + C content of LMG 1529^T and SKU 1109 is 62.5 and 63.3 mol% respectively. The name Gluconacetobacter maltaceti sp. nov. is proposed. The type strain is LMG 1529^T (= NBRC 14815^T = NCIMB 8752^T).     

Halomonas borealis sp. nov. and Halomonas niordiana sp. nov., two new species isolated from seawater

Two Gram-negative strains obtained from tank water in a scallop hatchery in Norway, were phenotypically and genotypically characterized in order to clarify their taxonomic position. On the basis of 16S rRNA gene sequence analysis, these isolates, ATF 5.2^T and ATF 5.4^T, were included in the genus Halomonas, being their closest relatives H. smyrnensis and H. taeanensis, with similarities of 98.9% and 97.7%, respectively. Sequence analysis of the housekeeping genes atpA, ftsZ, gyrA, gyrB, mreB, rpoB, rpoD, rpoE, rpoH, rpoN and rpoS clearly differentiated the isolates from the currently described Halomonas species, and the phylogenetic analysis using concatenated sequences of these genes located them in two robust and independent branches. DNA–DNA hybridization (eDDH) percentage, together with average nucleotide identity (ANI), were calculated using the complete genome sequences of the strains, and demonstrate that the isolates constitute two new species of Halomonas, for which the names of Halomonas borealis sp. nov. and Halomonas niordiana sp. nov. are proposed, with type strains ATF 5.2^T (=CECT 9780^T = LMG 31367^T) and ATF 5.4^T (=CECT 9779^T = LMG 31227^T), respectively.     

Vibrio aestivus sp. nov. and Vibrio quintilis sp. nov., related to Marisflavi and Gazogenes clades,respectively

Two new Vibrio species, Vibrio aestivus and Vibrio quintilis, are described after a polyphasic characterization of strains M22^T, M61 and M62^T, isolated from seawater collected off a beach on the East coast of Spain (Valencia). All three strains are Gram negative, mesophilic, slightly halophilic, fermentative rods. V. aestivus (M22^T = CECT 7558^T = CAIM 1861^T = KCTC 23860^T and M61 = CECT 7559 = CAIM 1862 = KCTC 23861) is oxidase positive, reduces nitrates to nitrites, is negative for Voges Proskauer, arginine dihydrolase and indole and non hydrolytic on most substrates tested. The 16S rRNA gene sequences of M22^T and M61 are most similar to Vibrio marisflavi (97.1–97.2%) but phylogenetic analysis using NJ, MP and ML methods display Vibrio stylophorae (96.2% similarity) as sibling species. The three species form a deep clade in the genus Vibrio. Average Nucleotide Identity (ANI) values, determined as a measure of overall genomic resemblance, confirmed that strains M22^T and M61 are members of the same species, different to V. marisflavi CECT 7928^T.V. quintilis (M62^T = CECT 7734^T = CAIM 1863^T = KCTC 23833^T) is aerogenic, arginine dihydrolase and Voges Proskauer positive, oxidase negative and unable to reduce nitrate, traits shared by most species in the Gazogenes clade. It is unpigmented and does not grow on TCBS Agar. 16S rRNA gene similarities to its nearest species, Vibrio aerogenes and Vibrio mangrovi, are 97.6% and 96.0% respectively. Strain M62^T and V. aerogenes CECT 7868^T display ANI values well below the 95% boundary for genomic species.