Taxogenomics of the Genus Cyclobacterium: Cyclobacterium xiamenense and Cyclobacterium halophilum as Synonyms and Description of Cyclobacterium plantarum sp. nov.

microorganisms Article Taxogenomics of the Genus Cyclobacterium: Cyclobacterium xiamenense and Cyclobacterium halophilum as Synonyms and Description of Cyclobacterium plantarum sp. nov. Azadeh Shahinpei 1, Mohammad Ali Amoozegar 1, Leila Mirfeizi 1, Mahdi Moshtaghi Nikou 2, Antonio Ventosa 3,* and Cristina Sánchez-Porro 3 1Extremophiles Laboratory, Department of Microbiology, Faculty of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, 1417414418 Tehran, Iran; [email protected] (A.S.); [email protected] (M.A.A.); [email protected] (L.M.) 2Microorganisms Bank, Iranian Biological Resource Centre (IBRC), ACECR, 1551916111 Tehran, Iran; [email protected] 3Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain; [email protected] *Correspondence: [email protected]; Tel.: +34-954-556-765 Received: 5 April 2020; Accepted: 20 April 2020; Published: 23 April 2020   Abstract: The genus Cyclobacterium belongs to the phylum Bacteroidetes and includes eight species. Our study, based on the genomic parameters in silico DNA–DNA hybridization (GGDC), average nucleotide identity (OrthoANI), and average amino acid identity (AAI), confirmed that all current species of Cyclobacterium belong to this genus and constitute a coherent phylogenomic group, but with species forming two separate branches. In addition, the genome-based analyses revealed that Cyclobacterium xiamenense and Cyclobacterium halophilum are members of the same species. Besides, we carried out a taxonomic characterization of the new strain GBPx2 T , isolated from the halophytic plant Salicornia sp. Analysis of its 16S rRNA gene sequence showed the highest sequence similarity (97.5%) to Cyclobacterium lianum HY9 T . Percentages of GGDC and OrthoANI between strain GBPx2 T and species of the genus Cyclobacterium were lower than the threshold value for species delineation. The DNA G+C content was 43.0 mol%. The polar lipids included phosphatidylethanolamine as well as one unidentified phospholipid and four unidentified lipids, and its major cellular fatty acids were iso-C 15:0 and summed feature 3 (C 16:1ω 7c and/or iso-C 15:0 2-OH). The only quinone present was menaquinone 7. Based on a combination of phenotypic, chemotaxonomic, and phylogenomic features, the GBPx2 T strain represents a novel species of the genus Cyclobacterium, for which the name Cyclobacterium plantarum sp. nov. is proposed. The type strain of Cyclobacterium plantarum is GBPx2 T (=IBRC-M 10634T=LMG 28551T). Keywords: Cyclobacterium;Cyclobacterium xiamenense;Cyclobacterium halophilum;Cyclobacterium plantarum; taxogenomics; bacterial taxonomy; halophilic bacteria; new species 1. Introduction The genus Cyclobacterium is the type genus of the family Cyclobacteriaceae, order Cytophagales, within the class Cytophagia, in the phylum Bacteroidetes [ 1 ]. This genus was originally described by Raj and Maloy [ 2 ] and has been emended three times [ 3 – 5 ]. Cyclobacterium marinum (type species) was initially described as Microcyclus marinus (referring to its vibrioid bacterial cell morphology that exhibits a closed ring-like morphology) [ 6 ], and was reclassified as Flectobacillus marinus [ 7 ]. Microorganisms 2020,8, 610; doi:10.3390/microorganisms8040610 www.mdpi.com/journal/microorganisms Microorganisms 2020,8, 610 2 of 16 Recently, the genus Cyclobacterium has been established as a separate genus and species from the genus Flectobacillus, which includes its freshwater counterparts [ 2 ]. The genus Cyclobacterium comprises eight species with valid published names: Cyclobacterium marinum (type species) [ 2 ], Cyclobacterium amurskyense [ 8 ], Cyclobacterium lianum [ 3 ], Cyclobacterium qasimii [ 9 ], Cyclobacterium caenipelagi [ 4 ], Cyclobacterium jeungdonense [ 10 ], Cyclobacterium xiamemense [ 5 ], and Cyclobacterium halophilum [ 11 ]. There is also another species, “Cyclobacerium sediminis”, which was described in 2017, but its name has not been validated to date [ 12 ]. These species have been isolated from different saline habitats such as seawater, marine sediments, soil from solar salterns, aggregates of the alga Chlorella autotrophica, or from sea cucumber [ 2 – 5 , 8 – 11 ]. Furthermore, 16S rRNA gene sequences related to this genus have also been reported by culture-dependent and/or culture-independent studies in different habitats such as a soda saline crater lake [ 13 ], microbial mats from Antarctic lakes [ 14 ], or from an alkaline, cold habitat in Greenland [ 15 ]. The species of this genus include Gram-stain-negative, curved ring-like or horseshoe-shaped bacteria. These species are non-flagellated, non-motile, aerobic, and heterotrophic, and their colonies are pigmented pink to orange/red. The major or sole respiratory quinone is MK-7. They are psychrotolerant to mesophilic and halotolerant to moderately halophilic. The G+C content of their DNA ranges from 33.7 to 48.1 mol% and their major cellular fatty acids (>10%) are iso-C 15:0 and summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c) [2–5,8–11]. In 2010, strain GBPx2 T was isolated from Salicornia sp., a halophytic plant, in the Gomishan wetland, Iran. This strain was affiliated to the genus Cyclobacterium but differed in some phenotypic and phylogenetic features from the Cyclobacterium species. We propose that it represents a novel species of the genus Cyclobacterium. Genome-based analysis was performed, using the genome sequences of the type strains of the species of Cyclobacterium and those of species of the family Cyclobacteriaceae available in databases, in order to carry out a taxogenomic study and determine in detail the phylogenomic relationships among species of the genus Cyclobacterium and other members of the family. 2. Materials and Methods 2.1. Bacterial Strains Strain GBPx2 T was isolated from Salicornia sp., a halophytic plant of the Gomishan wetland, an alkaline, thalassohaline, coastal-marine wetland located along the eastern shore of the Caspian Sea in Iran. The wetland water contains 30–50 g dissolved salts per liter, and the average pH is 8.8 [ 16 ]. The geographic coordinates of the sampling location were 37 ◦ 03 0 64.2 00 N 054 ◦ 01 0 90.4 00 E. Plants were sampled from southeastern the wetland in November 2010. The novel strain was isolated from a halophytic plant by using serial dilutions: 10 g of the sample was weighed and added to 90 mL of sterile 3.0% (w/v) NaCl solution. Serial dilutions were plated on Marine Agar 2216 (MA; Difco) and incubated at 30 ◦ C for two weeks. The colony of the strain was subsequently purified three times by plating on the same medium. It was maintained on the same medium and also at − 80 ◦ C in MA medium without agar and supplemented with 37.8% (w/v) glycerol. The type strains of three Cyclobacterium species were obtained from the Iranian Biological Resource Center (IBRC) and used as reference strains. These were Cyclobacterium lianum IBRC-M 10422 T , Cyclobacterium jeungdonense IBRC-M 11102 T , and Cyclobacterium halophilum IBRC-M 10761 T . They were cultured following the recommendations of the culture collection. 2.2. Taxophylogenomic Characterization 2.2.1. DNA Extraction, Purification, and Sequencing DNA was extracted following the protocol of Marmur [ 17 ]. The DNA quality was checked by (0.8%) agarose gel electrophoresis. The quantification of the extracted DNA was determined by spectrophotometry (DeNovix DS-11 FX, DeNovix Technologies, Wilmington, DA, USA) and fluorometry (Qubit 3.0 Fluorometer, Thermofisher Scientific, Waltham, MA, USA). PCR products were purified using Microorganisms 2020,8, 610 3 of 16 the commercial kit MEGAquick-spinTM Plus (INtRON Biotecnology, Labotaq, Sevilla, Spain). Sequencing of the 16S rRNA PCR products was carried out by Macrogene (Sangdaewon-dong, Gyeonggi-do, South Korea) using the Sanger method and the primers 16F27 (5 0 -AGAGTTTGATCMTGGCTCAG-3 0 ), 16R343 (5 0 -ACTGCTGCCTCCCGTA-3 0 ), 16F530 (5 0 -GTGCCAGCAGCCGCGG-3 0 ), and 16R1488 (5 0 -CGGTTACCTTGTTAGGACTTCACC-3 0 ) [ 18 ], and the genome of strain GBPx2 T was sequenced using the Illumina NovaSeq 6000 platform (Novogene Europe, Cambridge, UK). 2.2.2. Phylogenetic Analysis Based on 16S rRNA Gene Sequence Comparison The partial 16S rRNA gene was amplified using the universal primer pairs 16F27 and 16R1488 [ 19 ]. The PCR products were visualized on 1% agarose gel. The forward and reverse sequences were assembled by using Chromas Pro 1.7.7 (Technelysium Pty Ltd., South Brisbane, Australia). The 16S rRNA gene sequence of strain GBPx2 T was obtained and used for BLAST searches in GenBank and phylogenetic analysis. The identification of phylogenetic neighbors and calculation of pairwise 16S rRNA gene sequence similarity were achieved using the EzBioCloud server (https://www.ezbiocloud. net/) [ 20 ] and the alignments were performed by CLUSTAL-X [ 21 ]. Evolutionary distances between aligned 16S rRNA gene sequences of strain GBPx2 T with the most closely related type strains were calculated using the Jukes–Cantor model, and phylogenetic trees were reconstructed by the neighbour-joining [ 22 ], minimum-evolution [ 23 ], and maximum-likelihood [ 24 ] methods using the MEGA version 6 program [ 25 ]. Bootstrap analysis was carried out to evaluate the tree topology by performing resampling 1000 times [ 26 ]. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GBPx2 T is MG457806. The 16S rRNA gene sequences of the reference type strains used for the phylogenetic comparison were obtained from GenBank database and their accession numbers are shown in Figure 1. 2.2.3. Genome Assembly and Annotation The novo assembly of the reads of the genome of strain GBPx2 T was performed using Spades 3.13.0 [ 27 ]. The quality of final contigs was assessed by bioinformatics tools CheckM v1.0.5 [ 28 ] and Quast v2.3 [ 29 ]. The genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [ 30 ]. The genome of strain GBPx2 T was deposited in GenBank/EMBL/DDBJ under the accession number JAANYN00000000. 2.2.4. Phylogenomic Comparative Analysis For the phylogenomic comparative analysis we used genomes available from GenBank database. The characteristics of the genomes and their accession numbers of the type strains of species of the genus Cyclobacterium are shown in Table 1. The quality of these genome sequences was in accordance with the recommended minimal standards for the use of genome data for the taxonomy of prokaryotes [ 31 ]. To determine the core-genome, the Enveomics [32] tool was used. To identify clusters of orthologous genes (OGs), an all-versus-all BLAST search based on protein-coding gene annotated sequences of strain GBPx2 T and all type species of the genera included in the family Cyclobacteriaceae available in databases was carried out. Those OGs shared among all taxa and present in a single copy per genome were selected. They were aligned with MUSCLE v. 3.8.31 [ 33 ] and subsequently concatenated. A maximum-likelihood tree was constructed using FastTree v. 2.1.9 [ 34 ] with the JTT replacement matrix [ 35 ] under the CAT approximation (single rate for each site) with 20 rate categories. Local support values were estimated with the Shimodaira–Hasegawa test [36]. Microorganisms 2020,8, 610 4 of 16 Table 1. General features of the genomes of the type strains of species of the genus Cyclobacterium. Feature 1 2 3 4 5 6 7 8 Size (bp) 6,169,285 6,158,829 5,662,104 5,792,371 5,675,162 6,221,270 6,291,928 5,784,474 Contigs 37 1 30 41 31 1 202 98 Genome coverage 193X 101X 175X 100X 247X 30X 240X 100X G+C(mol%) 43.0 38.3 48.4 44.0 45.5 38.1 38.8 48.5 N50 (bp) 547,880 6,158,829 350,204 381,560 266,214 6,221,273 107,474 137,064 Total genes 4943 4833 4689 4646 4736 4981 5997 4595 Protein coding genes 4818 4715 4635 4534 4687 4868 5958 4474 rRNA 6 12 5 7 5 9 4 7 tRNA 41 39 38 40 38 39 35 39 Accession number JAANYN000000000 CP012040 FNZH00000000 WMCD00000000 FRCY00000000 NC_015914 ATNM00000000 WIOK00000000 Strains: 1, Strain GBPx2 T ; 2, Cyclobacterium amurskyense KCTC 12363 T ; 3, Cyclobacterium halophilum IBRC-M 10761 T ; 4, Cyclobacterium jeungdinense KCTC 23150 T ; 5, Cyclobacterium lianum CGMCC 1.6102T; 6, Cyclobacterium marinum DSM 745T; 7, Cyclobacterium qasimii M12-11BT; 8, Cyclobacterium xiamenense CGMCC 1.12432T. Microorganisms 2020,8, 610 5 of 16 2.2.5. In Silico DNA–DNA Hybridization (GGDC), Average Nucleotide Identity (ANI), and Average Amino Acid Identity (AAI) The genomic parameters of in silico DNA–DNA hybridization (GGDC), average nucleotide identity (OrthoANI), and average amino acid identity (AAI) among strain GBPx2 T , the type strains of species of the genus Cyclobacterium, and the type species of the family Cyclobacteriaceae available from databases were determined. GGDC was calculated by the bioinformatic tool Genome-to-Genome Distance Calculator (GGDC version 2.1) available from the Leibniz Institute DSMZ [ 37 ]. The OrthoANI was calculated with ChunLab’s Orthologous Average Nucleotide Identity Tool (OAT) [ 38 ]. For the estimation of the AAI, the CompareM program (https://github.com/dparks1134/CompareM) was used. 2.3. Phenotypic Characterization Cell morphology and motility were examined using an Olympus BX51 microscope equipped with phase-contrast optics with cells from exponentially growing cultures. Gram staining was performed by the Burke method [ 39 ]. Motility was determined by the wet-mount method [ 39 ]. Colony morphology was observed on MA agar medium under optimal growth conditions after incubation at 25 ◦ C for two days. To determine the temperature and pH ranges for growth, broth cultures of MA medium were incubated at 0, 4, 10, 15, 20, 25–37 (at intervals of 1.0 ◦ C), 40, and 45 ◦ C and at pH 5–10 at intervals of 0.5 pH units; the buffers sodium acetate/acetic acid (pH 5.0–6.0), Tris/HCl (pH 6.5–8.5), and glycine/sodium hydroxide (pH 9.0–10.0) were added at a concentration of 50 mM. The requirements for NaCl for growth were determined in media containing 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.5, 10.0, 12.5, and 15.0% (w/v) NaCl. Liquid cultures were incubated on a shaking incubator at 150 rpm and growth rates were determined by monitoring the increase in the optical density (OD) at 600 nm (ThermoSpectronics Spectronic 20D+). Catalase and oxidase tests, nitrate and nitrite reduction, hydrolysis of aesculin, and production of indole and H 2 S were carried out as recommended by Smibert and Krieg [ 40 ], using media with 5% (w/v) NaCl. Hydrolysis activity of Tween 20, 40, and 80 was detected as described by Guti é rrez and Gonz á lez [ 41 ]. Hydrolysis of gelatin, casein, tyrosine, and starch, and activity for urease and DNase were determined as described by Mata et al. [ 42 ]. The anaerobic growth of the strain was tested in the presence of nitrate by adding 0.1% (w/v) KNO 3 to the medium with 5% (w/v) NaCl in filled stoppered tubes in an anaerobic chamber [ 43 ]. Acid production from carbohydrates was tested in unbuffered medium and was determined by measuring the initial and final pH of the medium. The culture was considered positive for acid production if the pH decreased by at least 1 unit. Tests for the utilization of different compounds as the sole source of carbon and energy were performed as recommended by Ventosa et al. [44]. 2.4. Antimicrobial Susceptibility Antimicrobial susceptibility tests were performed on Mueller–Hinton agar plus 5% (w/v) marine salts [ 44 ] seeded with a bacterial suspension (in 5% [w/v] salts) containing 1.5 × 10 6 c.f.u. mL −1 using discs (HiMedia) impregnated with various antimicrobial compounds. The plates were incubated at 25 ◦ C for 48 h and the inhibition zone was interpreted according to the manufacturer’s manual. The following antimicrobial compounds were used: amoxicillin (30 µ g), ampicillin (10 µ g), bacitracin (10 µ g), carbenicillin (100 µ g), cefradine (30 µ g), ceftazidime (30 µ g), cephalothin (30 µ g), chloramphenicol (30 µ g), erythromycin (15 µ g), gentamicin (10 µ g), kanamycin (5 µ g), nalidixic acid (30 µ g), neomycin (30 µ g), nitrofurantoin (300 µ g), novobiocin (5 µ g), penicillin G (10 U) polymyxin B (300 U), rifampicin (5 µg), streptomycin (10 µg), and tetracycline (30 µg). 2.5. Chemotaxonomic Characterization Cell biomass for fatty acids, isoprenoid quinones, and polar lipids analyses was obtained by cultivation on MA medium at pH 8 and 25 ◦ C. Cells were harvested in the mid-exponential growth Microorganisms 2020,8, 610 6 of 16 phase determined spectrophotometrically with an optical density at 600 nm (OD 600 ). The whole-cell fatty acids composition of strain GBPx2 T was determined according to the standard protocol of the Microbial Identification System (MIDI, Version 6.1; Identification Library TSBA40 4.1; Microbial ID). Extracts were analyzed using a Hewlett Packard model HP6890A gas chromatograph equipped with a flame-ionization detector as described by Kämpfer and Kroppenstedt [ 45 ]. Fatty acids peaks were identified using the TSBA40 database. The polar lipids and respiratory quinones of strain GBPx2 T were analyzed as described by Groth et al. [46]. 3. Results and Discussion 3.1. Phylogenetic Analysis Based on 16S rRNA Gene Sequence Comparison The 16S rRNA gene sequence comparative analysis of strain GBPx2 T (1438 nt) showed the highest similarity to Cyclobacterium lianum HY9 T ,Cyclobacterium jeungdonense HMD3055 T ,Cyclobacterium xiamenense KD51 T , and Cyclobacterium halophilum IBRC-M 10761 T with 97.5%, 96.7%, 96.2%, and 96.2% sequence similarity, respectively, and values lower than 92.3% with species of other genera, such as Belliella or Fontibacter. These percentages were obtained by the EzBioCloud tool and indicate that strain GBPx2Tis a member of the genus Cyclobacterium. The 16S rRNA gene sequence phylogenetic analysis using the maximum-likelihood algorithm showed the position of the novel strain within the genus Cyclobacterium (Figure 1). The phylogenetic position was also confirmed in trees generated using the minimum-evolution and neighbour-joining algorithms. Microorganisms 2020, 8, x FOR PEER REVIEW 6 of 16 Figure 1. Maximum-likelihood phylogenetic tree based on the 16S rRNA gene sequence comparison, showing the relationships between strain GBPx2T and members of the family Cyclobacteriaceae. Filled circles indicate nodes that were also obtained in trees based on minimum-evolution and maximumlikelihood algorithms. Bootstrap values (for 1000 replicates) over 70% are shown at the nodes. The sequence accession numbers are shown in parenthesis. Bar, 2% estimated sequence divergence. The sequence of Parapedobacter soli DCY14T (EF151805) was used as outgroup. This phylogenetic tree shows that the genus Cyclobacterium is not monophyletic; the species of this genus are grouped into two clearly differentiated branches supported with 100% values of bootstrap. On the one hand, C. xiamenense KD51T, C. halophilum GASx41T, C. jeungdonense HMD3055T, C. lianum HY9T, and the new isolate GBPx2T appear grouped, and on the other hand C. marinum LMG 13164T, C. qasimii M12-11BT, C. caenipelagi HD-17T, and C. amurskyense KMM 6143T are clustered. To determine the relationship between these two clusters, a phylogenomic comparative analysis between them and also with members of other genera of the family Cyclobacteriaceae was performed. 3.2. Phylogenomic Comparative Analysis We carried out phylogenomic comparative analysis and obtained the core-genome tree, based on 1309 single-copy translated genes of strain GBPx2T, the genomes available for the type strains of Cyclobacterium species (Table 1), and the genomes of all type species of the genera of the family Cyclobacteriaceae available in databases (Figure 2). This analysis shows that strain GBPx2T constitutes a taxon which is sufficiently different from the other species of Cyclobacterium so as to be considered as a new species. Further, as occurred in the phylogenetic tree based on the 16S rRNA, the species of the genus Cyclobacterium appeared grouped in two different branches. Finally, this phylogenomic tree showed a close phylogenetic relationship between Cyclobacterium xiamenense CGMCC 1.12432T and Cyclobacterium halophilum IBRC-M 10761T, two species that were described almost simultaneously in 2014 [5,11], and so they were not considered for a comparison between them. Besides, the genomes of these two species are only now available for comparison and the current comparative data show in this study revealed that both are members of the same species. Figure 1. Maximum-likelihood phylogenetic tree based on the 16S rRNA gene sequence comparison, showing the relationships between strain GBPx2 T and members of the family Cyclobacteriaceae. Filled circles indicate nodes that were also obtained in trees based on minimum-evolution and maximum-likelihood algorithms. Bootstrap values (for 1000 replicates) over 70% are shown at the nodes. The sequence accession numbers are shown in parenthesis. Bar, 2% estimated sequence divergence. The sequence of Parapedobacter soli DCY14T(EF151805) was used as outgroup. This phylogenetic tree shows that the genus Cyclobacterium is not monophyletic; the species of this genus are grouped into two clearly differentiated branches supported with 100% values of bootstrap. On the one hand, C. xiamenense KD51 T ,C. halophilum GASx41 T ,C. jeungdonense HMD3055 T ,C. lianum HY9 T , and the new isolate GBPx2 T appear grouped, and on the other hand C. marinum LMG 13164 T , Microorganisms 2020,8, 610 7 of 16 C. qasimii M12-11B T ,C. caenipelagi HD-17 T , and C. amurskyense KMM 6143 T are clustered. To determine the relationship between these two clusters, a phylogenomic comparative analysis between them and also with members of other genera of the family Cyclobacteriaceae was performed. 3.2. Phylogenomic Comparative Analysis We carried out phylogenomic comparative analysis and obtained the core-genome tree, based on 1309 single-copy translated genes of strain GBPx2 T , the genomes available for the type strains of Cyclobacterium species (Table 1), and the genomes of all type species of the genera of the family Cyclobacteriaceae available in databases (Figure 2). This analysis shows that strain GBPx2 T constitutes a taxon which is sufficiently different from the other species of Cyclobacterium so as to be considered as a new species. Further, as occurred in the phylogenetic tree based on the 16S rRNA, the species of the genus Cyclobacterium appeared grouped in two different branches. Finally, this phylogenomic tree showed a close phylogenetic relationship between Cyclobacterium xiamenense CGMCC 1.12432 T and Cyclobacterium halophilum IBRC-M 10761 T , two species that were described almost simultaneously in 2014 [ 5 , 11 ], and so they were not considered for a comparison between them. Besides, the genomes of these two species are only now available for comparison and the current comparative data show in this study revealed that both are members of the same species. Microorganisms 2020, 8, x FOR PEER REVIEW 7 of 16 Figure 2. Phylogenomic tree based on the core orthologous translated genes of strain GBPx2T, type species of Cyclobacterium, and type species of the genera of the family Cyclobacteriaceae obtained from the genomes available in databases, based on the maximum-likelihood algorithm. This tree was obtained after the alignment of 1309 shared orthologous single-copy translated genes of these genomes. Bootstrap values higher than 70% are indicated at branch-points. Bar, 0.1 substitutions per amino acid position. 3.3. in silico DNA–DNA Hybridization (GGDC), ANI, and AAI Values In order to confirm that strain GBPx2T was indeed a new taxon and the relationship between C. xiamenense and C. halophilum and the two clusters of the genus Cyclobacterium, average nucleotide identity (OrthoANI), average amino acid identity (AAI), and in silico DNA–DNA hybridization (GGDC) for the strain GBPx2T and members of the family Cyclobacteriaceae were calculated. GGDC percentages above or equal to 70% indicate that the strains can be assigned to the same species, and values under 70% indicate that the strains belong to different species [47–49]. GGDC values were equal or lower than 35% between strain GBPx2T and species of the genus Cyclobacterium (Table 2), proving that this strain constitutes a new species. In addition, the GGDC value of 81.6% which was determined between C. xiamenense CGMCC 1.12432T and C. halophilum IBRC-M 10761T, which was higher than the threshold percentage of 70% for species delineation, shows that both species belong to the same taxon [31,37]. With respect to the GGDC values between the other members of this family, all were lower than 70%, showing that all of them can be considered different taxa at the species level. OrthoANI percentages calculated between strain GBPx2T and species of the genus Cyclobacterium ranged from 71.8% to 79.2% (Table 2), lower than the threshold value for species delineation (95%– 96%) [31,38,49,50], showing that strain GBPx2T belongs to a different species. Values between 67.2% and 69.9% with the type species of the other genera of the family Cyclobacteriaceae were obtained. Further, the OrthoANI value of 97.8% between C. xiamenense CGMCC 1.12432T and C. halophilum IBRC-M 10761T showed again that both species constituted a single taxon. An alternative to GGDC and ANI for more distantly related genomes is the AAI. In this case, to confirm that strain GBPx2T and all species of Cyclobacterium were well assigned to this genus, the AAI percentages between them were calculated. The AAI values between each other were in the range of 72.2%–97.9% (Table 3). These values were above the threshold considered for species of the same Pleomorphovibrio marinusSW125T(QCXY01000001) Lunatimonas lonarensis AK24T(AQHR00000000) Echinicola pacifica DSM 19836T(ARDN00000000) Algoriphagusratkowskyi DSM 22686T(MSSV00000000) Belliella baltica DSM 15883T(CP003281) Rhodonellum psychrophilum GCM71T(ARMB00000000) Indibacter alkaliphilus LW1T(ALWO00000000) Mongoliibacter ruber DSM 27929T(PRJNA402522) Cecembia lonarensisLW9T(AMGM00000000) MariniradiussaccharolyticusAK6T (AMZY0000000) Aquiflexum balticum DSM 16537T(LT838813) Nitritalea halalkaliphila LW7T(AJYA00000000) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 0.10 Cyclobacterium jeungdonese KCTC 12363T(WMCD00000000) Cyclobacterium halophilum IBRC-M 10761T(FNZH00000000) Cyclobacterium xiamenense CGMCC 1.12432T(WIOK00000000) Cyclobacterium lianum CGMCC 1.6102T(FRCY00000000) Cyclobacterium plantarum GBPx2T (JAANYN000000000) Cyclobacterium marinum DSM 745T(NC_015914) Cyclobacterium amurskyense KCTC 12363T (CP_012040) Cyclobacterium quasimii M12-11BT(ATNM00000000) Figure 2. Phylogenomic tree based on the core orthologous translated genes of strain GBPx2 T , type species of Cyclobacterium, and type species of the genera of the family Cyclobacteriaceae obtained from the genomes available in databases, based on the maximum-likelihood algorithm. This tree was obtained after the alignment of 1309 shared orthologous single-copy translated genes of these genomes. Bootstrap values higher than 70% are indicated at branch-points. Bar, 0.1 substitutions per amino acid position. 3.3. in silico DNA–DNA Hybridization (GGDC), ANI, and AAI Values In order to confirm that strain GBPx2 T was indeed a new taxon and the relationship between C. xiamenense and C. halophilum and the two clusters of the genus Cyclobacterium, average nucleotide identity (OrthoANI), average amino acid identity (AAI), and in silico DNA–DNA hybridization (GGDC) for the strain GBPx2Tand members of the family Cyclobacteriaceae were calculated. Microorganisms 2020,8, 610 8 of 16 GGDC percentages above or equal to 70% indicate that the strains can be assigned to the same species, and values under 70% indicate that the strains belong to different species [ 47 – 49 ]. GGDC values were equal or lower than 35% between strain GBPx2 T and species of the genus Cyclobacterium (Table 2), proving that this strain constitutes a new species. In addition, the GGDC value of 81.6% which was determined between C. xiamenense CGMCC 1.12432 T and C. halophilum IBRC-M 10761 T , which was higher than the threshold percentage of 70% for species delineation, shows that both species belong to the same taxon [ 31 , 37 ]. With respect to the GGDC values between the other members of this family, all were lower than 70%, showing that all of them can be considered different taxa at the species level. OrthoANI percentages calculated between strain GBPx2 T and species of the genus Cyclobacterium ranged from 71.8% to 79.2% (Table 2), lower than the threshold value for species delineation (95%–96%) [ 31 , 38 , 49 , 50 ], showing that strain GBPx2 T belongs to a different species. Values between 67.2% and 69.9% with the type species of the other genera of the family Cyclobacteriaceae were obtained. Further, the OrthoANI value of 97.8% between C. xiamenense CGMCC 1.12432 T and C. halophilum IBRC-M 10761Tshowed again that both species constituted a single taxon. An alternative to GGDC and ANI for more distantly related genomes is the AAI. In this case, to confirm that strain GBPx2 T and all species of Cyclobacterium were well assigned to this genus, the AAI percentages between them were calculated. The AAI values between each other were in the range of 72.2%–97.9% (Table 3). These values were above the threshold considered for species of the same genus (65%) [ 50 – 52 ], so we can affirm that all species belong to the genus Cyclobacterium. It is remarkable to highlight that AAI values between C. marinum DSM 745 T ,C. qasimii M12-11B T , and C. amurskyense KMM 6143 T were higher (83.5% to 87.5%) as compared to with other species of Cyclobacterium (72.2%–73.5%), and lower than 66.2% with respect to species of the rest of genera of the family Cyclobacteriaceae. Similar results were observed in the other group of species of the genus Cyclobacterium that appear grouped in the 16S rRNA phylogenetic tree (Figure 1) and also in the core-genome tree (Figure 2). This group included C. xiamenense KD51 T ,C. halophilum IBRC-M 10761 T ,C. jeungdonense HMD3055 T ,C. lianum CGMCC 1.6102 T , and the new isolate GBPx2 T . AAI values between them ranged from 77.2% to 73.5%. With respect to the other species of the genus Cyclobacterium the AAI ranged between 72.2% and 73.5%, and values ranged between 61.6% to 68.1% with regard to the rest of the genera of the family Cyclobacteriaceae. All these data showed that the percentages for species of Cyclobacterium were always higher than 65% and thus they are members of the same genus, although there was a higher similarity between the respective members of the two phylogroups. Therefore, we conclude that the genus Cyclobacterium is monophyletic within the family, but once differentiated, it is divided into two clearly separated groups, as observed previously in both the 16S rRNA and core-genome trees (Figures 1and 2). On the other hand, the value of 97.9% confirms that C. xiamenense and C. halophilum are members of the same taxon, as was described by Konstantinidis et al. [ 51 ] who established the threshold AAI range of 95%–100% for strains of the same species. Microorganisms 2020,8, 610 9 of 16 Table 2. Percentages of GGDC and OrthoANI between strain GBPx2Tand members of the family Cyclobacteriaceae. Microorganisms 2020, 8, x FOR PEER REVIEW 9 of 16 Table 2. Percentages of GGDC and OrthoANI between strain GBPx2T and members of the family Cyclobacteriaceae. Percentages of similarity 100 90–99 80–89 70–79 60–69 50–59 40–49 30–39 20–29 10–19 0–9 OrthoANI 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 100 78.1 80.0 71.8 71.4 71.2 70.6 70.5 69.3 68.5 68.5 68.2 69.7 68.8 68.7 69.0 68.4 67.8 69.1 67.8 2 26.9 100 82.4 72.0 71.1 71.4 70.6 70.5 68.8 68.1 68.3 68.4 69.6 68.6 68.7 69.0 68.6 67.7 68.9 67.6 3 31.3 44.4 100 72.1 71.1 71.5 70.7 70.6 69.0 68.2 68.2 68.1 69.3 68.8 68.7 69.0 68.6 67.6 69.1 67.6 4 14.3 14.6 14.5 100 79.2 76.0 74.3 74.1 69.9 69.1 68.4 67.7 69.0 69.0 68.7 68.7 68.9 68.0 69.0 67.5 5 14.1 13.8 14.1 35.0 100 74.1 73.5 73.4 69.6 69.2 68.0 67.4 69.3 68.6 68.2 68.4 68.5 67.9 68.9 67.2 6 14.2 14.3 14.4 20.8 17.8 100 76.6 76.4 69.3 69.2 68.0 67.5 68.6 68.8 68.3 68.2 68.7 67.9 68.7 67.3 7 13.9 13.9 14 17.4 16.9 24.2 100 97.8 69.4 69.3 68.1 67.1 68.1 68.3 67.9 67.9 68.2 68.0 68.5 67.6 8 14.0 13.9 14.1 17.4 16.7 23.9 81.6 100 69.2 69.2 68.0 67.1 68.3 68.2 68.1 68.2 68.1 67.7 68.7 67.6 9 12.9 12.8 12.8 13 13 12.9 12.9 12.9 100 69.6 67.3 67.2 68.5 68.5 68.6 68.4 68.7 67.7 69.0 67.1 10 12.9 12.8 12.9 12.9 12.8 12.9 13.1 13.0 13.1 100 68.0 67.4 68.8 68.3 68.9 68.3 69.2 68.1 68.9 67.8 11 12.7 12.8 12.8 12.7 12.7 12.7 12.7 12.7 12.7 12.7 100 68.7 69.5 68.6 68.9 69.0 69.0 68.3 69.0 68.1 12 12.7 12.7 12.7 12.6 12.5 12.6 12.6 12.6 12.6 12.6 12.7 100 69.7 69.0 68.7 68.9 68.6 68.0 69.0 67.5 13 12.8 12.8 12.9 12.8 12.9 12.8 12.7 12.7 12.8 12.8 12.8 13.0 100 71.7 72.5 71.7 71.5 69.7 72.4 69.3 14 12.8 12.8 12.8 12.7 12.7 12.7 12.7 12.7 12.7 12.7 12.8 12.8 13.6 100 70.9 70.7 70.8 70.0 71.7 68.1 15 12.8 12.8 12.8 12.8 12.8 12.7 12.8 12.8 12.7 12.9 12.7 12.8 14.1 13.4 100 75.7 72.7 70.1 72.5 68.4 16 12.8 12.7 12.8 12.7 12.7 12.7 12.8 12.8 12.7 12.7 12.9 12.8 13.6 13.2 21.2 100 72.2 69.9 72.4 68.2 17 12.7 12.7 12.7 12.7 12.7 12.8 12.7 12.7 12.8 12.8 12.9 12.7 13.4 13.2 14.5 14.1 100 70.6 72.5 69.1 18 12.7 12.7 12.6 12.7 12.6 12.7 12.7 12.6 12.7 12.7 12.8 12.7 12.9 12.9 13.1 13.2 12.8 100 70.9 68.1 19 12.8 12.7 12.7 12.8 12.9 12.7 12.7 12.7 12.8 12.8 12.8 12.7 13.6 13.2 13.8 13.8 13.9 13.3 100 68.4 20 12.7 12.7 12.7 12.7 12.6 12.7 12.7 12.7 12.6 12.7 12.8 12.7 13.1 12.8 12.8 12.8 13.1 12.7 12.8 100 Strains: 1, Cyclobacterium marinum DSM 745T; 2, Cyclobacterium qasimii M12-11BT; 3, Cyclobacterium amurskyense KCTC 12363T; 4, Strain GBPx2T; 5, Cyclobacterium lianum CGMCC 1.6102T; 6, Cyclobacterium jeungdinense KCTC 23150T; 7, Cyclobacterium xiamenense CGMCC 1.12432T; 8, Cyclobacterium halophilum IBRC-M 10761T; 9, Pleomorphovibrio marinus SW125T; 10, Lunatimonas lonarensis AK24T; 11, Echinicola pacifica DSM 19836T; 12, Algoriphagus ratkowskyi DSM 22686T; 13, Belliella baltica DSM 15883T; 14, Rhodonellum psychrophilum GCM71T; 15, Indibacter alkaliphilus LW1T; 16, Mongoliibacter ruber DSM 27929T; 17, Cecembia lonarensis LW9T; 18, Mariniradius saccharolyticus AK6T; 19, Aquiflexum balticum DSM 16537T; 20, Nitritalea halalkaliphila LW7T. ANI: average nucleotide identity. GGDC Strains: 1, Cyclobacterium marinum DSM 745 T ; 2, Cyclobacterium qasimii M12-11B T ; 3, Cyclobacterium amurskyense KCTC 12363 T ; 4, Strain GBPx2 T ; 5, Cyclobacterium lianum CGMCC 1.6102 T ; 6, Cyclobacterium jeungdinense KCTC 23150 T ; 7, Cyclobacterium xiamenense CGMCC 1.12432 T ; 8, Cyclobacterium halophilum IBRC-M 10761 T ; 9, Pleomorphovibrio marinus SW125 T ; 10, Lunatimonas lonarensis AK24 T ; 11, Echinicola pacifica DSM 19836 T ; 12, Algoriphagus ratkowskyi DSM 22686 T ; 13, Belliella baltica DSM 15883 T ; 14, Rhodonellum psychrophilum GCM71 T ; 15, Indibacter alkaliphilus LW1 T ; 16, Mongoliibacter ruber DSM 27929 T ; 17, Cecembia lonarensis LW9 T ; 18, Mariniradius saccharolyticus AK6 T ; 19, Aquiflexum balticum DSM 16537 T ; 20, Nitritalea halalkaliphila LW7T. ANI: average nucleotide identity. Microorganisms 2020,8, 610 16 of 16 47. Kim, M.; Oh, H.S.; Park, S.C.; Chun, J. 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