Vahid Keshavarz-Tohid

Jordan Vacheron

Audrey Dubost

Claire Prigent-Combaret

Parissa Taheri

Seyed Saeed Tarighi

Mohsen Taghavi

Yvan Moenne-Loccoz

Daniel Muller

Pseudomonas persica sp. nov Pseudomonas Shirazica

Sp Nov

Vahid Keshavarz-Tohid

Seyed Mohsen Taghavi

Yvan Moënne-Loccoz

Genomic, phylogenetic and catabolic re-assessment of the Pseudomonas putida clade supports the delineation of Pseudomonas alloputida sp. nov., Pseudomonas

Keywords: nov., Pseudomonas persica sp. nov., and Pseudomonas shirazica sp. nov

published or not. The documents may come

Introduction

Pseudomonas is one of the most complex and diverse bacterial genera [START_REF] Palleroni | The Pseudomonas story[END_REF][START_REF] Peix | The current status on the taxonomy of Pseudomonas revisited: An update[END_REF], encompassing over 250 described species as of May 2018 [START_REF] Euzéby | List of bacterial names with standing in nomenclature: a folder available on the internet[END_REF][START_REF] Parte | LPSN -List of prokaryotic names with standing in nomenclature (bacterio.net), 20 years on[END_REF] (http://www.bacterio.net/pseudomonas.html). Most species from this genus seem ubiquitous and were isolated from a variety of distinctive habitats in water, soil, and eukaryotic hosts [START_REF] Silby | Pseudomonas genomes: diverse and adaptable[END_REF].

Although some species or strains were shown to be pathogenic for humans [START_REF] De Bentzmann | The Pseudomonas aeruginosa opportunistic pathogen and human infections[END_REF], animals [START_REF] Bergan | Human-and animal-pathogenic members of the genus Pseudomonas, in the prokaryotes: a handbook on habitats, isolation, and identification of bacteria[END_REF][START_REF] López | Pseudomonas baetica sp. nov., a fish pathogen isolated from wedge sole, Dicologlossa cuneata (Moreau)[END_REF][START_REF] Nishimori | Pseudomonas plecoglossicida sp. nov., the causative agent of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis[END_REF] or plants [START_REF] Xin | Pseudomonas syringae pv. tomato DC3000: a model pathogen for probing disease susceptibility and hormone signaling in plants[END_REF][START_REF] Patel | Identification of virulence associated loci in the emerging broad host range plant pathogen Pseudomonas fuscovaginae[END_REF], most Pseudomonas genotypes are inoffensive as commensal members of the microbiota [START_REF] Peix | The current status on the taxonomy of Pseudomonas revisited: An update[END_REF] or even beneficial to their eukaryotic hosts (e.g. plant growth-promoting rhizobacteria [START_REF] Haas | Biological control of soil-borne pathogens by fluorescent pseudomonads[END_REF][START_REF] Ramette | Pseudomonas protegens sp nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin[END_REF][START_REF] Almario | Rhizosphere ecology and phytoprotection in soils naturally suppressive to Thielaviopsis black root rot of tobacco[END_REF]). Thus, this bacterial taxon presents a large variety of lifestyles, plays diverse roles in biochemical cycles [START_REF] Richardson | Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms[END_REF][START_REF] Peix | Pseudomonas rhizosphaerae sp. nov., a novel species that actively solubilizes phosphate in vitro[END_REF], and produces various metabolites of biotechnological interest [START_REF] Puchałka | Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology[END_REF], such as vitamin B12 [START_REF] Kang | Recent advances in microbial production of δ-aminolevulinic acid and vitamin B12[END_REF], siderophores [START_REF] Cornelis | Iron uptake and metabolism in pseudomonads[END_REF], antibiotics [START_REF] Almario | Rhizosphere ecology and phytoprotection in soils naturally suppressive to Thielaviopsis black root rot of tobacco[END_REF][START_REF] Loper | Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions[END_REF][START_REF] Hesse | Genome-based evolutionary history of Pseudomonas spp[END_REF][START_REF] Weller | Pseudomonas biocontrol agents of soilborne pathogens: looking back over 30 years[END_REF] or phytohormones [START_REF] Loper | Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions[END_REF][START_REF] Hesse | Genome-based evolutionary history of Pseudomonas spp[END_REF][START_REF] Flury | Insect pathogenicity in plant-beneficial pseudomonads: phylogenetic distribution and comparative genomics[END_REF].

Although species might emerge through ecological specialization [START_REF] Lassalle | Ecological speciation in bacteria: reverse ecology approaches reveal the adaptive part of bacterial cladogenesis[END_REF][START_REF] Lassalle | Ancestral genome estimation reveals the history of ecological diversification in Agrobacterium[END_REF], current bacterial species definition relies on molecular (genomic) homogeneity between strains of the species. In brief, microbial species delineation relies on a polyphasic approach [START_REF] Wayne | Report of the ad hoc committee on reconciliation of approaches to bacterial systematics[END_REF][START_REF] Stackebrandt | Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology[END_REF] based originally on (i) the change in melting temperature (or ΔTm) of heteroduplex DNA formed upon annealing of the DNAs from pairwise-tested strains, and (ii) DNA-DNA hybridization (DDH) percentage [START_REF] Wayne | Report of the ad hoc committee on reconciliation of approaches to bacterial systematics[END_REF][START_REF] Stackebrandt | Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology[END_REF] or whole genome sequence identity computed as average nucleotide identity (ANI) [START_REF] Li | The relationship of the whole genome sequence identity to DNA hybridization varies between genera of prokaryotes[END_REF]. In addition, sequence comparison of DNA taxonomic markers (house-keeping genes such as rrs, gyrB or rpoD) and characterization of phenotypic traits (morphological, biochemical and/or enzymatic properties) are combined to assemble a set of strains in a species [START_REF] Wayne | Report of the ad hoc committee on reconciliation of approaches to bacterial systematics[END_REF][START_REF] Stackebrandt | Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology[END_REF].

Over 70 new Pseudomonas species have been described in the last ten years [START_REF] Peix | The current status on the taxonomy of Pseudomonas revisited: An update[END_REF], and recent analyses based on ANI calculations are suggesting the existence of several uncharacterized species (or genomospecies [START_REF] Tran | In-silico taxonomic classification of 373 genomes reveals species misidentification and new genospecies within the genus Pseudomonas[END_REF]). Based on multilocus sequence analyses (MLSA) with rrs, gyrB, rpoD and rpoB and ANI-based genome comparisons, the Pseudomonas genus is divided in three main lineages, each subdivided in several phylogenetic groups : the P.

fluorescens lineage is constituted of 7 groups, the P. aeruginosa lineage 3 groups and the P.

pertucinogena lineage 1 group [START_REF] Peix | The current status on the taxonomy of Pseudomonas revisited: An update[END_REF][START_REF] Mulet | DNA sequence-based analysis of the Pseudomonas species[END_REF][START_REF] García-Valdés | Pseudomonas: Molecular phylogeny and current taxonomy[END_REF].

Within the P. fluorescens lineage, the P. putida group is the second largest one in the number of described species [START_REF] Garrido-Sanz | Classification of isolates from the Pseudomonas fluorescens complex into phylogenomic groups based in group-specific markers[END_REF]. These species are mainly studied for their biotechnological potential [START_REF] Poblete-Castro | Industrial biotechnology of Pseudomonas putida and related species[END_REF], in relation to the production of particular chemicals [START_REF] Schmid | Industrial biocatalysis today and tomorrow[END_REF][START_REF] Wackett | Pseudomonas putida-a versatile biocatalyst[END_REF][START_REF] Ward | A Two Step Chemo-biotechnological conversion of polystyrene to a biodegradable thermoplastic[END_REF] or phytobeneficial properties [START_REF] Vacheron | Fluorescent Pseudomonas strains with only few plant-beneficial properties are favored in the maize rhizosphere[END_REF][START_REF] Agaras | Genomic insights into the broad antifungal activity, plant-probiotic properties, and their regulation, in Pseudomonas donghuensis strain SVBP6[END_REF]. P. putida was isolated in 1889 [START_REF] Migula | Über ein neues System der Bakterien[END_REF][START_REF] Trevisan | I generi e le specie delle Batteriacee[END_REF], and since then other species were isolated from clinical samples (P. mosselii [START_REF] Dabboussi | Pseudomonas mosselii sp. nov., a novel species isolated from clinical specimens[END_REF] and P. monteilii [START_REF] Elomari | Pseudomonas monteilii sp. nov., isolated from clinical specimens[END_REF][START_REF] Molina | Specific gene loci of clinical Pseudomonas putida isolates[END_REF]), infected animals (P.

entomophila L48 [START_REF] Mulet | Taxonomic characterisation of Pseudomonas strain L48 and formal proposal of Pseudomonas entomophila sp[END_REF] and P. plecoglossicida [START_REF] Nishimori | Pseudomonas plecoglossicida sp. nov., the causative agent of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis[END_REF]), soil (P. soli [START_REF] Pascual | Pseudomonas soli sp. nov., a novel producer of xantholysin congeners[END_REF], P. vranovensis [START_REF] Tvrzová | Pseudomonas moraviensis sp. nov. and Pseudomonas vranovensis sp. nov., soil bacteria isolated on nitroaromatic compounds, and emended description of Pseudomonas asplenii[END_REF], P.

taiwanensis [START_REF] Wang | Pseudomonas taiwanensis sp. nov., isolated from soil[END_REF], P. fulva, P. parafulva, P. cremoricolorata [START_REF] Uchino | Recharacterization of Pseudomonas fulva Iizuka and Komagata 1963, and proposals of Pseudomonas parafulva sp. nov. and Pseudomonas cremoricolorata sp[END_REF], and P. guariconensis [START_REF] Toro | Pseudomonas guariconensis sp. nov., isolated from rhizospheric soil[END_REF]) and water (P. donghuensis [START_REF] Gao | Pseudomonas donghuensis sp. nov., exhibiting high-yields of siderophore[END_REF][START_REF] Yonezuka | Phylogenetic analysis reveals the taxonomically diverse distribution of the Pseudomonas putida group[END_REF]). Many isolates of the P. putida group have been classified as P.

putida strains based on 16S rRNA gene homology. However, most of them do not belong to the P. putida species sensu stricto, but to genomospecies within the P. putida group [START_REF] Yonezuka | Phylogenetic analysis reveals the taxonomically diverse distribution of the Pseudomonas putida group[END_REF][START_REF] Mulet | Phylogenetic affiliation of Pseudomonas putida biovar A and B strains[END_REF][START_REF] Gomila | Phylogenomics and systematics in Pseudomonas[END_REF].

Recently, Keshavarz-Tohid et al. [START_REF] Keshavarz-Tohid | Phylogenetic diversity and antagonistic traits of root and rhizosphere pseudomonads of bean from Iran for controlling Rhizoctonia solani[END_REF] isolated bacteria from the bean rhizosphere, and 18 of the isolates were affiliated to the P. putida group based on phylogenetic analysis of taxonomic markers. These isolates were distributed over five phylogenetic clusters (termed Pp1 to Pp5) that did not include type strains, raising the possibility that they might represent new species within the P. putida group.

The objective of this work was to clarify whether the Pseudomonas isolates of Keshavarz-Tohid et al. [START_REF] Keshavarz-Tohid | Phylogenetic diversity and antagonistic traits of root and rhizosphere pseudomonads of bean from Iran for controlling Rhizoctonia solani[END_REF] could correspond to new species, and if so to establish these new species in taxonomic terms. To this end, we inferred the phylogeny of the isolates and other strains of the P. putida group, sequenced the genome of some of them, and compared in silico these bacteria to the type strains from the P. putida group and a large panel of strains published as P. putida (we will hereafter refer to them as P. 'putida' in cases where they were misnamed as P. putida) and for which the genomic sequence was available. Thus, the phylogeny of 95 strains of the P. putida group was investigated by MLSA, and they were assessed based on average nucleotide identity (ANI), digital DNA hybridizations and phenotyping. This resulted in the proposition of four new species within the P. putida group, i.e. P. alloputida sp. nov., P.

inefficax sp. nov., P. persica sp. nov. and P. shirazica sp. nov.

Material and methods

Bacterial strains, culture conditions and DNA extraction

Bacterial strains were previously isolated from different soils from Iran or France and affiliated to the Pseudomonas genus [START_REF] Vacheron | Fluorescent Pseudomonas strains with only few plant-beneficial properties are favored in the maize rhizosphere[END_REF][START_REF] Keshavarz-Tohid | Phylogenetic diversity and antagonistic traits of root and rhizosphere pseudomonads of bean from Iran for controlling Rhizoctonia solani[END_REF]. Briefly, 20 strains originated from rhizosphere of bean plants from three Iranian provinces: strains RUB1 (previously VF16) and RUB2

(previously VF13) from Fars province; VKh2, VKh4, VKh9, VKh10, RUB6 (previously VKh13), VKh7, VKh14, VKh17 from Khorasan province ; VM2, VM3, VM10, VM11, VM13, VM14 and RUB5 (previously VM6) from Mazandaran province [START_REF] Keshavarz-Tohid | Phylogenetic diversity and antagonistic traits of root and rhizosphere pseudomonads of bean from Iran for controlling Rhizoctonia solani[END_REF]. Three strains named JV241A, JV551A1 and JV551A3 were isolated from the rhizosphere of maize grown in French soil (Béligneux, 30 km East from Lyon) [START_REF] Vacheron | Fluorescent Pseudomonas strains with only few plant-beneficial properties are favored in the maize rhizosphere[END_REF]. Besides this collection, P. 'monteilii' SB3078 [START_REF] Dueholm | Complete genome sequences of Pseudomonas monteilii SB3078 and SB3101, two benzene-, toluene-, and ethylbenzene-degrading bacteria used for bioaugmentation[END_REF] (hereafter renamed P. shirazica), P. 'putida' W15Oct28 [START_REF] Ye | Draft genome sequence analysis of a Pseudomonas putida W15Oct28 strain with antagonistic activity to Gram-positive and Pseudomonas sp. pathogens[END_REF], P. 'putida' BW11M1 [START_REF] Ghequire | Draft genome sequence of Pseudomonas putida BW11M1, a banana rhizosphere isolate with a diversified antimicrobial armamentarium[END_REF] (hereafter renamed P. mosselii) and P. cremoricolorata DSM 17059 T [START_REF] Ye | Draft genome sequence analysis of a Pseudomonas putida W15Oct28 strain with antagonistic activity to Gram-positive and Pseudomonas sp. pathogens[END_REF] were obtained from Aalborg University (Denmark), Université Libre de Bruxelles (Belgium), Catholic University Leuven (Belgium) and University of Malaya (Malaysia), respectively. The 18 strains were routinely cultivated overnight on King's B [START_REF] King | Two simple media for the demonstration of pyocyanin and fluorescin[END_REF] or LB [START_REF] Bertani | The mode of phage liberation by lysogenic Escherichia coli[END_REF] media at 28°C with shaking (150 rpm). Bacterial genomic DNA was extracted from 500 μL of bacterial culture using the NucleoSpin Tissue kit (Macherey-Nagel, Hoerdt, France), following the manufacturer's instructions. DNA was quantified spectrophotometrically and adjusted to 30 ng μL -1 .

Phylogenetic analysis

The phylogenetic analyses included 126 Pseudomonas strains with complete or draft genome sequences (accession numbers available in Table S1), 13 Pseudomonas strains with sequenced taxonomic markers (Table S2; see below), and Cellvibrio japonicus Ueda 107 T as outgroup. Nucleotidic sequences were retrieved from Genbank (Table S1) and aligned using MUSCLE v3.8.31 [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF]. Alignments were used to compute Maximum Likelihood trees using PhyML [START_REF] Guindon | New algorithms and methods to estimate Maximum-Likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF], with 500 bootstraps, and SeaView v4 [START_REF] Gouy | SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF]. Phylogenetic trees from individual rrs (16S rRNA gene), gyrB and rpoB sequences were generated (not shown), as well as a phylogenetic tree based on concatenated sequences with a total length of 2963 nucleotides (1436 for rrs, 820 for gyrB, 707 for rpoD).

Phenotypic profiling

Strains RUB1, VKh7, RUB6, VKh14, VM14, JV551A1, JV551A3, P. 'monteilii' SB3078, P. 'putida' W15Oct28, P. 'putida' BW11M1 and P. cremoricolorata DSM 17059 T were tested for phenotypic characteristics, and results compared with published data on the other strains. Cell morphology and number of flagella were investigated by using the method of Heimbrook et al. [START_REF] Heimbrook | Staining bacterial flagella easily[END_REF]. Biochemical tests were done with Biolog GEN III MicroPlate (BIOLOG, Hayward, CA), according to manufacturer's instructions, using 71 carbon sources and 23 chemical sensitivity assays over 48 h. All tests were done two times.

Genome sequencing

As described above, genomic DNA was extracted from strains JV241A, JV551A1, JV551A3, RUB1, RUB6, VM14, VKh7 and VKh14 grown overnight in King's B [START_REF] King | Two simple media for the demonstration of pyocyanin and fluorescin[END_REF]. The resulting DNA samples were sent to Molecular Research LP (Shallowater, TX, USA), where library preparation was performed using the Nextera DNA sample preparation kit (Illumina Inc., San Diego, CA, USA). Genomic DNA was then sequenced using Illumina MiSeq systems and assembled using SeqMan NGen® version 12.0 (DNASTAR, Madison, WI, USA) with pairedend sequencing parameters on the default settings. Genomeannotation was done with the online MicroScope platform [START_REF] Vallenet | MicroScope-an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data[END_REF]. The draft genome sequences can be found under bioprojects PRJEB24813, PRJEB24814, PRJEB24815, PRJEB25064, PRJEB25066, PRJEB25068, PRJEB25065, PRJEB25067 (Table S1).

Computation of average nucleotide identities and digital DNA-DNA hybridizations

Average Nucleotide Identity (ANI) was calculated for 126 complete or draft genomes with fastANI [START_REF] Jain | High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[END_REF] (Table S3 and Figure 1). In total, the dataset consisted of 15,750 pairwise values (excluding the 126 pairwise comparisons of each genome with itself). The Genome-to-Genome Distance Calculator GGDC 2.1 [START_REF] Meier-Kolthoff | Genome sequence-based species delimitation with confidence intervals and improved distance functions[END_REF][START_REF] Meier-Kolthoff | Complete genome sequence of DSM 30083T, the type strain (U5/41T) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy[END_REF][START_REF] Meier-Kolthoff | Taxonomic use of DNA G+C content and DNA-DNA hybridization in the genomic age[END_REF] was used to calculate the digital DNA-DNA hybridization (dDDH) estimates between genomes of candidate strains, so as to define new species in comparison with genomes of type strains from the P. putida group.

Results

Phylogenetic classification of strains affiliated to the P. putida group

A phylogenetic tree from concatenated rrs, gyrB and rpoD genes was generated for all 126 strains (Figure 2). In addition to 17 type strains and our 20 isolates of the P. putida group, the tree contains strains from the P. putida group for which the sequenced genome is available in a database (some of them shown here to be misnamed as P. putida), and a set of representative type strains of the P. fluorescens group and the P. aeruginosa lineage. The different strains affiliated so far to P. putida were retrieved in different Pseudomonas clades, with P. 'putida'

FDAARGOS121 branching within the P. aeruginosa lineage, and P. 'putida' CBB5 and MC4 5222 within the P. fluorescens group.

Only 3 of 65 non-type strains that were confirmed here to belong to the P. putida group did correspond to the P. putida species (Figure 2). None of our 20 isolates [START_REF] Vacheron | Fluorescent Pseudomonas strains with only few plant-beneficial properties are favored in the maize rhizosphere[END_REF][START_REF] Keshavarz-Tohid | Phylogenetic diversity and antagonistic traits of root and rhizosphere pseudomonads of bean from Iran for controlling Rhizoctonia solani[END_REF] belonged to P. putida species; three of them were found close to type strains, i.e. P. wadenswilersensis CCOS 864 T [START_REF] Frasson | Pseudomonas wadenswilerensis sp. nov. and Pseudomonas reidholzensis sp. nov., two novel species within the Pseudomonas putida group isolated from forest soil[END_REF] for strain JVA241A (96% sequence identity for the concatenated rrs, gyrB and rpoD sequences) and P. soli LMG27941 T for cluster-Pp1 strains RUB1 and RUB2 (each with 98% identity). The remaining 17 strains were distributed in four distinct clusters (Pp2, Pp3, Pp4, Pp5) or close to cluster Pp3 (for strain JV551). Although clusters Pp2, Pp3, Pp4 and Pp5 include various sequenced bacteria named P. putida or P. monteilii so far, none of them contain the P.

putida type strain (or any other type strain). Based on previous use of multilocus sequence analyses to identify putative new Pseudomonas species [START_REF] Gomila | Phylogenomics and systematics in Pseudomonas[END_REF][START_REF] Mulet | Concordance between whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry and multilocus sequence analysis approaches in species discrimination within the genus Pseudomonas[END_REF], our findings point to the occurrence of four if not five new species in the P. putida group, and hereafter we define four proposed species i.e. P. shirazica (corresponding to cluster Pp4), P. alloputida (for cluster Pp5), P. persica (for cluster Pp2) and P. inefficax (for French strains of the Lyon area).

Genome sequence-based species delimitation

ANIs calculated using whole genome data for the eight strains sequenced and the 117 sequenced Pseudomonas strains retrieved (including 19 type strains) pointed to 33 genomic species (including the 19 species already described), based on ANI values ≥ 95% (Figure 1 and Table S3). The ANI values strengthened the proposal of the new species P. shirazica, P. alloputida, P. persica and P. inefficax, in that strains within each of them displayed ANIs above 95% with one another but that were below the 95% threshold with all other related strains tested (including type strains). This was the case for (i) P. 'putida' S16, P. 'monteilii' SB3078 and SB3101, and strains VM14, SF1, DLL-E4, S11, HB3267, MO2 and KG4 (now P. shirazica), (ii)

P. 'putida' BIRD-1, S12, PCL1760, YKD221, SJTE-1, DOT-TIE, PDI, TRO1, JCM 9802, B6-2,
INSali382, IDAHO, KT2440 and JLR11, and strains VKh7 (CFBP8484) and VKh14 (now P. alloputida), (iii) strain RUB6 (now P. persica), and (iv) strains JV551A1 and JV551A3 (now P. inefficax). Although the ANI value was below the 95% threshold (i.e. 94.9%) when comparing the RUB1 genome (5.5 Mb) with the partially-sequenced genome (only 0.9 Mb released) of P.

soli LMG 27941 T , the ANI was > 96% when comparing RUB1 to P. soli CCOS 191, suggesting that RUB1 is a new strain of the P. soli species. The difference in genome sequence availability had probably biased the ANI estimate with the type strain. As the genome sequence of P.

wadenswilerensis CCOS 864 T (the closest to JV241A) has not been released, it is not possible to clarify whether JV241A belongs to P. wadenswilerensis or to another, yet-undescribed species.

In addition to ANI comparisons, the dDDH values determined using all type strains (with GGDC 2.1 [START_REF] Meier-Kolthoff | Genome sequence-based species delimitation with confidence intervals and improved distance functions[END_REF][START_REF] Meier-Kolthoff | Complete genome sequence of DSM 30083T, the type strain (U5/41T) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy[END_REF][START_REF] Meier-Kolthoff | Taxonomic use of DNA G+C content and DNA-DNA hybridization in the genomic age[END_REF]) showed that for strains VKH7 (proposed type strain for P. alloputida), VM14 (proposed type strain for P. shirazica), JV551A1 (proposed type strain for P. inefficax), P. 'putida' W15Oct28 and RUB6 (proposed type strain for P. persica), the values were all significantly lower than the cut-off of 70% (Table S4). This confirms, together with ANI, that the five strains are representatives of novel species in the P. putida group. dDDH values calculated with other sequenced strains from the P. putida group indicated that two of the proposed new species contain several of these strains, which so far have been incorrectly affiliated to other species (P. putida or P. monteilii ; see Table S4). It is the case for (i) strains LS46, TRO1, B6-2, YKD221, ND6, F1, JLR11, KT2440, INSali382, DOT-T1E, S12, S12_GCF, BIRD-1, VKh14, IOFA19, PCL1760, PD1, SJTE-1, IOFA1, LF54, H, Idaho and JCM 9802, whose dDDH value with P. alloputida VkH7 (type strain) ranges from 70.6% (for TRO1) to ~100% (for VKh14 ;

Table S4), and (ii) strains S16, SB3078, SB3101, SF1, DLL-E4, S11, HB3267, MO2 and KG4, whose dDDH value with P. shirazica VM14 (type strain) ranges from 78.8% (for S16) to 89.2%

(for HB3267).

Morphological and biochemical features

When grown on King's B agar, P. soli RUB1, P. alloputida VkH7 (proposed type strain), P. persica RUB6, VKh14, P. shirazica VM14 (proposed type strain), P. inefficax JV551A1 (proposed type strain), P. inefficax JV551A3, P. shirazica SB3078, P. 'putida' W15Oct28, P. mosselii BW11M1 and P. cremoricolorata DSM 17059 T formed circular, convex colonies producing fluorescent pigment(s). All were Gram-negative, aerobic and rod shaped.

Table 1 shows the phenotypic characteristics differentiating the 14 type strains of the P.

putida group (a more complete set of data is presented in Table S5). In the proposed species P.

alloputida, strains VKh7 (proposed type strain), KT2440 and VKh14 presented negative results (GEN III MicroPlate, Biolog) for D-melibiose, p-hydroxy-phenylacetic acid, -keto-butyric acid and ß-methyl-D-glucoside tests, whereas its closest relatives P. putida and P. monteilli were positive for the same tests (Table 1). In the proposed species P. persica, strain RUB6 (proposed type strain) gave negative results for bromo-succinic acid, D-galacturonic acid, D-glucuronic acid, D-mannose and p-hydroxy-phenylacetic acid tests and positive results for acetic acid, Dserine, glucuronamide and sucrose tests, in contrast to its closest relatives P. guariconenesis and P. inefficax. In the proposed species P. inefficax, strain JV551A3 (proposed type strain) displayed positive results for bromo-succinic acid, D-galacturonic acid, D-glucuronic acid, Dmannose and p-hydroxy-phenylacetic acid tests. In addition, P. inefficax JV551A1 and JV551A3 differed from P. alloputida VKh7 and VKh14 in that their formic acid tests were positive and their sucrose tests negative. In the proposed species P. shirazica, strain VM14 (proposed type strain) gave a positive result in the formic acid test and negative results for N-acetyl-Dglucosamine, D-galacturonic acid, L-galactonic acid lactone and D-glucuronic acid tests.

For P. 'putida' W15Oct28, particular morphological and phenotypic characteristics were found. In contrast to members of related species P. putida and P. fulva, strain W15Oct28 could not grow at 37°C and harbored 2 or 3 polar flagella. Contrarily to P. alloputida VKh7 and VKh14, strain W15Oct28 gave a positive result for p-hydroxy-phenylacetic acid test and negative results for sucrose, D-mannose and D-galacturonic acid tests. Taken together, genomic and phenotypic data are suggesting that strain W15Oct28 is a member of a new genomic species.

Discussion

Members of the P. putida group play important ecological roles in various ecosystems, especially in soils and sediments, and have received considerable research attention for plant growth promotion, biodegradation of organic contaminants, and other biotechnological usages [START_REF] Wu | Comparative genomics and functional analysis of niche-specific adaptation in Pseudomonas putida[END_REF][START_REF] Loeschcke | Pseudomonas putida-a versatile host for the production of natural products[END_REF]. The prominent strain KT2440 (i.e. mt-2 / DSM 6125 / ATCC 47054) alone (cluster Pp5; reclassified as P. alloputida in this work) has been the focus of several hundred publications.

In 2011 [START_REF] Wu | Comparative genomics and functional analysis of niche-specific adaptation in Pseudomonas putida[END_REF], genomes of strains F1, KT2440, W619 and GB-1 were compared as member of a same species, but in the present work we demonstrated that strains F1 and KT2440 are members of the P. alloputida species (cluster Pp5), whereas GB-1 (close to cluster Pp5) and W619 (branching between P. reidholzensis CCOS 865 T and P. taiwanensis BCRC 17751 T ) are strains representing two new genomic species. Moreover, GB-1 represents the closest genomic species to P. alloputida and strain W619 together with strain ATH43 form another genomospecies more distantly related to P. putida since several other species are branching between W619 and P. putida sensu stricto (see Figure 2 and Table S3). Interestingly, Wu et al. [START_REF] Wu | Comparative genomics and functional analysis of niche-specific adaptation in Pseudomonas putida[END_REF] noticed that W619 presented the most different genome architecture (synteny around the replication origin is not conserved with the other strains) and heavy metal gene organization compared to P. alloputida strain KT2440 and strain F1 or to P. 'putida' GB-1. Similarly, Lidbury et al. [START_REF] Lidbury | Comparative genomic, proteomic and exoproteomic analyses of three Pseudomonas strains reveals novel insights into the phosphorus scavenging capabilities of soil bacteria[END_REF] analyzed the phosphorous scavenging capabilities of different Pseudomonas species and grouped strains in different species, with strains KT2440 and BIRD-1 together (now in P. alloputida), and GB-1 in a new genomic species and W619 in another new genomic species.

The taxonomy of P. putida and related species has long been recognized in need of a clarification [START_REF] Molina | Specific gene loci of clinical Pseudomonas putida isolates[END_REF][START_REF] Yonezuka | Phylogenetic analysis reveals the taxonomically diverse distribution of the Pseudomonas putida group[END_REF][START_REF] Hesse | Genome-based evolutionary history of Pseudomonas spp[END_REF], and it is in this context that the current taxonomic assessment was carried out. Comparative genomic analysis using all the genome sequences available in public databases enabled to define 33 genomic species encompassing the 19 already described species (Figures 1,2, Tables S3 andS4). The other 14 genomic species (currently unnamed) encompassed strain with incorrect species affiliations (Table 2). Four genomic species among the 14 were assessed by differential phenotypic analysis, which enabled to propose four new species.

Taxonomic characterization of the new isolates

Phylogenetic and genomic analyses showed that certain isolates could be classified as members of a new Pseudomonas species, and strain Kh7 (= LMG 29756 / CFBP 8484; cluster Pp5) was designated as the type strain for Pseudomonas alloputida sp. nov. (Table 3). The new species includes strain KT2440, which had been recognized as not belonging to the P. putida species [START_REF] Regenhardt | Pedigree and taxonomic credentials of Pseudomonas putida strain KT2440[END_REF]. Indeed, DNA-DNA hybridization between strains P. putida DSM 291 T and KT2440 was estimated to be 50.5% [START_REF] Regenhardt | Pedigree and taxonomic credentials of Pseudomonas putida strain KT2440[END_REF], i.e. below the 70% threshold that delimits bacterial species. The phenotypic characterization shows profiles (Tables 1 andS5) of all representative type strains of the Pseudomonas putida group. From the main characteristics, the P. alloputida species is characterized by Gram-negative cells forming white colonies on Kings' B medium, approximately 3 mm in diameter after 24 h growth. The cells were rods, motile and 1.6-5 μm in length, with a growth temperature optimum of 28°C and an optimum pH of 7.0. Their genome size ranged from 5.7 to 6.48 Mb with a GC% between 61.39% and 61.99% (Tables 4, S6 and S7).

Phenotypic and phylogenetic analyses enabled to propose strain RUB6 (= LMG 29757 / CFBP 8486; cluster Pp2) as the type strain of the new species Pseudomonas persica (detailed characteristics are given in Table 5). From the main characteristics, P. persica corresponds to Gram-negative cells forming white colonies on Kings' B medium, approximately 3 mm in diameter after 24 h of growth. Compared with neighboring (sister) species found in the MLSA tree (Figure 2), P. persica cannot use bromo-succinic acid, D-galacturonic acid, D-gluronic acid but was able to use sucrose (unique among the tested species of the P. putida group). The cells were rods, motile, and 1.6-5 μm in length, with an optimum growth temperature of 28°C and optimum pH of 7.0. Genome size was 5.42 Mb, with a GC% of 62.92% (Tables 4, S6 and S7).

Comparative genomic analysis enabled to propose Pseudomonas shirazica sp. nov, with VM14 (= LMG 29953 / CFBP 8487; within cluster Pp4) proposed as the type strain (Table 6). P.

shirazica encompasses strains that were isolated from various environments and that exhibit a variety of phenotypes. Indeed, strain VM14 was isolated from bean rhizosphere [START_REF] Keshavarz-Tohid | Phylogenetic diversity and antagonistic traits of root and rhizosphere pseudomonads of bean from Iran for controlling Rhizoctonia solani[END_REF], SB3078 and SB3101 degrade benzene, toluene, and ethylbenzene [START_REF] Dueholm | Complete genome sequences of Pseudomonas monteilii SB3078 and SB3101, two benzene-, toluene-, and ethylbenzene-degrading bacteria used for bioaugmentation[END_REF][START_REF] Dueholm | Survival and activity of individual bioaugmentation strains[END_REF], S16 degrades nicotine [START_REF] Wang | Characterization of environmentally friendly nicotine degradation by Pseudomonas putida biotype A strain S16[END_REF],

and HB3267 is a clinical isolate with high pathogenic potential [START_REF] Fernández | Analysis of the pathogenic potential of nosocomial Pseudomonas putida strains[END_REF]. P. shirazica is characterized by Gram-negative cells forming white colonies on Kings' B medium, approximately 3 mm in diameter after 24 h of growth. The cells are rods, motile, 1.6-5 μm in length, with a growth temperature optimum of 28°C and an optimum pH of 7.0. Genome size ranges from 5.51 to 6.48 Mb with a GC% between 61.96% and 63.02% (Tables 4 andS7).

The last newly described species encompasses two strains (JV551A3 and JV551A1;

between clusters Pp2 and Pp3 ; see Figure 2) isolated from maize rhizosphere [START_REF] Vacheron | Fluorescent Pseudomonas strains with only few plant-beneficial properties are favored in the maize rhizosphere[END_REF][START_REF] Vacheron | Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays[END_REF], with strain JV551A3 (= DSM 108619 / CFBP 8493) proposed as type strain for P. inefficax (Table 7).

None of the two strains presented any plant growth promotion effects when tested on maize or

Arabidopsis plants [START_REF] Vacheron | Fluorescent Pseudomonas strains with only few plant-beneficial properties are favored in the maize rhizosphere[END_REF][START_REF] Vacheron | Differential contribution of plant-beneficial functions from Pseudomonas kilonensis F113 to root system architecture alterations in Arabidopsis thaliana and Zea mays[END_REF]. Detailed species characteristics are summarized in Table 8. The cells are rods, motile, 1.6-5 μm in length, with a growth temperature optimum of 28°C and an optimum pH of 7.0. Genome size ranges from 6.42 to 5.88 Mb with a GC% between 62.85% and 62.75% (Tables 4, S6 and S7).

Figure legends

Figure 1 Genomic relationship between strains in the P. putida group based on ANI values (%), which were determined with fastANI [START_REF] Jain | High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[END_REF]. Type strains are in bold and the four new species in red. A more exhaustive comparison is provided in Table S2. Pseudomonas strains with sequenced taxonomic markers (blue dots). Cellvibrio japonicus Uedea 107 T was used as outgroup. In the figure, the groups and phylogenetic lineages proposed previously [START_REF] Peix | The current status on the taxonomy of Pseudomonas revisited: An update[END_REF][START_REF] Mulet | DNA sequence-based analysis of the Pseudomonas species[END_REF][START_REF] Hesse | Genome-based evolutionary history of Pseudomonas spp[END_REF] are indicated; type strains and proposed type strains are in bold. Within the Pseudomonas putida group, the Pseudomonas putida clusters proposed previously [START_REF] Keshavarz-Tohid | Phylogenetic diversity and antagonistic traits of root and rhizosphere pseudomonads of bean from Iran for controlling Rhizoctonia solani[END_REF] are highlighted in blue rectangles; the dotted lines are delimiting clusters of strains belonging to the same species according to ANI data. Concatenated sequences were used to compute the tree using PhyML [START_REF] Guindon | New algorithms and methods to estimate Maximum-Likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF], with 500 bootstraps, and SeaView v4 [START_REF] Gouy | SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF]. The tree was visualized using iTOL software [START_REF] Letunic | Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees[END_REF].

+ + + W - + - + + - + + D D-Arabitol - + + - - - - - - - - - - - D-Fructose D + + + W + + + - - + + + + D-Galacturonic acid - - - - - - + - - - - + + + D-Glucuronic acid D - - - - - + - - - + + + + D-Mannitol - + + + - - - - - - - - - - D-Mannose + + + + - - + + + + - + + + D-Melibiose + - - - - - - - - W - + + - D-Serine + + - - - + + D + + + + + D D-Sorbitol + - - - - - - - - - - - - - Dextrin + - - - - - - - - - - + + - Glucuronamide - - D - - + + + ND ND + + ND + L-Fucose - + W - - - - + - - - - - - L-
+ + + + + - - + - + + Sucrose - - - - - + - - - - - - - D Tween 40 - + + + + W D D + + - + + D α-Hydroxybutyric acid + D W D - - - - + + - D + - α-Keto-butyric acid D D + - - - D - + + - + - - β-Methyl-D-glucoside D D D D - - - - - W - w + - Formic acid + + + D - + + + + - - + + D
-, negative; + positive; ND, not done; W, weak; D, depends on the tested strain or experiment. a For Pseudomonas mosseli, data for type strain ATCC 700476 T [START_REF] Mulet | Taxonomic characterisation of Pseudomonas strain L48 and formal proposal of Pseudomonas entomophila sp[END_REF] and strain BW11M1 were combined.

b For P. soli, data for type strain F-279,208 T [START_REF] Pascual | Pseudomonas soli sp. nov., a novel producer of xantholysin congeners[END_REF] and strain F16 were combined. c For P. persica, data for type strain RUB6 T and strain VM16 were combined. d For P. inefficax, data for type strain JV551A3 T and strain JV551A1 were combined. e For P. shirazica, data for type strain VM13 T , strainVM14 and strain SB3078 were combined. f For P. alloputida, data for type strain VKh7 T , strain VKh14 and strain KT2440 were combined.

Submitter

Daniel MULLER E-mail of the submitter daniel.muller@univ-lyon1.fr

Designation of the type strain VKh7

Strain collection numbers CFBP 8484 = LMG 29756

16S rRNA gene accession number LT718459

Genome accession number [EMBL] PRJEB25065

Genome status Draft

Genome size 5707279

GC mol % 61.99

Country of origin Iran

Region of origin Khorasan Razavi province

Date of isolation

Number of strains in study 3

Source of isolation of non-type strains

Bean rhizosphere

Growth medium, incubation conditions

King's B agar (KBA) at 28°C

Gram stain Negative

Cell shape Rod

Motility Motile

If motile Flagellar

If flagellated Single polar

Sporulation (resting cells) None

Colony morphology

Positive tests with BIOLOG Aerobiosis

Negative tests with BIOLOG α-keto-glutaric acid, a-D-glucose, acetic acid, citric acid, Dfructose, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, D-mannose, D-saccharic acid, glucuronamide, glycerol, inosine, Lalanine, L-aspartic acid, L-glutamic acid, L-histidine, L-lactic acid, L-pyroglutamic acid, L-serine, propionic acid, quinic acid, γ-amino butyric acid, β-hydroxybutyric, aztreonam, vancomycin, guanidine HCl, lincomycin, minocycline, niaproof 4, rifamycin SV, tetrazolium blue, tetrazolium violet, troleandomycin, 1% NaCl, 1% sodium lactate, 4% NaCl, 8% NaCl, D-serine, D-fucose, fusidic acid, L-arginine, L-galactonic acid lactone, L-malic acid, lithium chloride, mucic acid, nalidixic acid, pH 5, pH 6, potassium tellurite, sodium bromate, sodium butyrate

Table 4

Genome size and GC% content of species in the P. putida group. For species that contain more than one sequenced strain, data are shown as means ± standard deviations. Raw data are shown in Table S6. 61.70 ± 0.19 a Strains for which the entire genome was not sequenced were not included (including the type strains of P. soli and P. guariconensis).

Species name

If motile Flagellar

If flagellated Single polar

Sporulation (resting cells) None

Colony morphology

Colonies were fluorescent, approximately 3 mm in diameter, circular and convex with an entire margin

Lowest temperature for growth 15

Highest temperature for growth 37 S3

Figure 2

 2 Figure 2 Maximum Likelihood phylogeny based on concatenated rrs-gyrB-rpoB genes of 138 Pseudomonas strains, i.e. 126 Pseudomonas strains with complete or draft genome sequences (indicated with red dots for genomes obtained during the current project) and 13

  Energy metabolism α-D-lactose, D-arabitol, D-cellobiose, D-galactose, D-glucose-6-PO 4 , D-mannitol, D-melibiose, D-raffinose, D-sorbitol, Dtrehalose, dextrin, gentiobiose, L-fucose, L-rhamnose, myoinositol, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, phydroxy-phenylacetic acid, turanose, α-hydroxybutyric acid, α-keto butyric acid, β-methyl-D-glucoside, gelatin, acetoacetic acid, pectin, 3-methyl glucose, D-aspartic acid, D-lactic acid methyl ester, D-malic acid, D-maltose, D-salicin, glycyl-L-proline, Nacetyl neuraminic acid, N-acetyl-ß-D-mannosamine, Variable tests with BIOLOG bromo-succinic acid, D-serine, methyl pyruvate, sucrose tween 40, formic acid Positive tests with API ADH, GLU-assim, MNE, GNT, CAP, MLT, CIT, PAC, OX Negative tests with API NO3, TRP, GLU_ Ferm, URE, ESC, GEL, PNPG, ARA, MAN, NAG, MAL, ADI Energy metabolism Chemoorganotroph

4 HighestLowest temperature for growth 4 Highest

 44 for strain testing Aerobiosis Positive tests with BIOLOG D-fructose, D-gluconic acid, D-saccharic acid, D-serine, glucuronamide, glycerol, inosine, L-alanine, L-aspartic acid, Lglutamic acid, L-histidine, L-pyroglutamic acid, L-serine, methyl pyruvate, propionic acid, quinic acid, sucrose, γ-amino butyric acid, β-hydroxybutyric, formic acid, vancomycin, guanidine HCl, lincomycin, minocycline, niaproof 4, rifamycin SV, tween 40, aztreonam, acetoacetic acid, tetrazolium blue, tetrazolium violet, troleandomycin,1% NaCl, 4% NaCl, 1% sodium lactate, D-serine, D-fucose, fusidic acid, L-arginine, L-malic acid, lithium chloride, mucic acid, nalidixic acid, pH 5, pH 6 Negative tests with BIOLOG Bromo-succinic acid, D-arabitol, D-cellobiose, D-galacturonic acid, D-glucose-6-PO 4 , D-glucuronic acid, D-mannitol, Dmannose, D-melibiose, D-raffinose, D-sorbitol, D-trehalose, dextrin, gentiobiose, L-fucose, L-rhamnose, N-acetyl-Dgalactosamine, N-acetyl-D-glucosamine, p-hydroxy-phenylacetic acid, turanose, α-hydroxybutyric acid, α-keto butyric acid, βmethyl-D-glucoside, gelatin, pectin, 3-methyl glucose, 8% NaCl, D-aspartic acid, D-lactic acid methyl ester, D-malic acid, Dmaltose, D-salicin, glycyl-L-proline, L-galactonic acid lactone, Nacetyl neuraminic acid, N-acetyl-ß-D-mannosamine, sodium bromate, sodium butyrate, stachyose, D-fructose-6Colonies were fluorescent, approximately 3 mm in diameter, circular and convex with an entire margin Lowest temperature for growth for strain testing Aerobiosis Positive tests with BIOLOG acetic acid, D-fructose, D-gluconic acid, D-mannose, D-saccharic acid, glucuronamide, glycerol, L-alanine, L-aspartic acid, L-fucose, L-glutamic acid, L-histidine, L-pyroglutamic acid, L-serine, methyl pyruvate, propionic acid, quinic acid, γ-amino butyric acid, βhydroxybutyric, formic acid, vancomycin, guanidine HCl, lincomycin, minocycline, niaproof 4, potassium tellurite, rifamycin SV, tetrazolium blue, tetrazolium violet, troleandomycin, 1% NaCl, 1% sodium lactate, 8% NaCl, D-fucose, L-arginine, L-malic acid, mucic acid, pH 5, pH 6 Negative tests with BIOLOG bromo-succinic acid, D-arabitol, D-cellobiose, D-galacturonic acid, D-glucose-6-PO 4 , D-glucuronic acid, D-mannitol, D-melibiose, Draffinose, D-sorbitol, D-trehalose, dextrin, gentiobiose, Lrhamnose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, phydroxy-phenylacetic acid, sucrose, turanose, α-hydroxybutyric acid, α-keto butyric acid, β-methyl-D-glucoside, gelatin, aztreonam, acetoacetic acid, pectin, 3-methyl glucose, D-serine, Daspartic acid, D-lactic acid methyl ester, D-malic acid, D-maltose, D-salicin, glycyl-L-proline, L-galactonic acid lactone, N-acetyl neuraminic acid, N-acetyl-ß-D-mannosamine, sodium bromate, stachyose, D-fructose-6-PO 4 Variable tests with BIOLOG D-serine, inosine, tween 40, 4% NaCl, fusidic acid nalidixic acid, sodium butyrate Colonies were fluorescent, approximately 3 mm in diameter, circular and convex with an entire margin for strain testing Aerobiosis Positive tests with BIOLOG D-arabitol, D-cellobiose, D-glucose-6-PO 4 , D-mannitol, D-melibiose, Daffinose, D-sorbitol, D-trehalose, dextrin, gentiobiose, L-fucose, L-rhamnose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, sucrose, turanose, αhydroxybutyric acid, β-methyl-D-glucoside, gelatin, acetoacetic acid, pectin, 3-methyl glucose, D-aspartic acid, D-lactic acid methyl ester, D-malic acid, D-maltose, D-salicin, glycyl-L-proline, N-acetyl neuraminic acid, N-acetyl-ß-D-mannosamine, stachyose, D-fructose-6-PO 4 Negative tests with BIOLOG acetic acid, bromo-succinic acid, D-fructose, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, D-mannose, D-saccharic acid, D-serine, glucuronamide, glycerol, inosine, L-alanine, L-aspartic acid, L-glutamic acid, L-histidine, L-pyroglutamic acid, L-serine, methyl

Figure 1

 1 Figure 1Genomic relationship between strains in the P. putida group based on ANI values (%), which were determined with fastANI[START_REF] Jain | High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[END_REF]. Type strains are in bold and the four new species in red. A more exhaustive comparison is provided in TableS3

Table 1

 1 Selected differential phenotypic characteristics of species of the P. putida group. Data are shown for the type strain or (when available) the mean data of type strain and related strains of the same species. Literature data are shown for P. plecoglossicida ATCC 700383 T[START_REF] Nishimori | Pseudomonas plecoglossicida sp. nov., the causative agent of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis[END_REF], P. guariconenesis PCAVU T[START_REF] Toro | Pseudomonas guariconensis sp. nov., isolated from rhizospheric soil[END_REF], P. parafulva DSM 17004 T[START_REF] Mulet | Taxonomic characterisation of Pseudomonas strain L48 and formal proposal of Pseudomonas entomophila sp[END_REF], P. fulva IAM 1529 T[START_REF] Mulet | Taxonomic characterisation of Pseudomonas strain L48 and formal proposal of Pseudomonas entomophila sp[END_REF], P. entomphila L48 T[START_REF] Mulet | Taxonomic characterisation of Pseudomonas strain L48 and formal proposal of Pseudomonas entomophila sp[END_REF], P. putida ATCC 12633 T[START_REF] Mulet | Taxonomic characterisation of Pseudomonas strain L48 and formal proposal of Pseudomonas entomophila sp[END_REF] and P. monteilii ATCC 700476 T[START_REF] Mulet | Taxonomic characterisation of Pseudomonas strain L48 and formal proposal of Pseudomonas entomophila sp[END_REF]. A full list of tested phenotypes is given in TableS5. Presented data were determined via GEN III MicroPlate (Biolog) metabolic tests. Type strains are in bold.

							T								T
		P. plecoglossicida ATCC	700383 T	P. entomphila L48 T	P. mosselii a	P. soli b	P. guariconenesis PCAVU	P. persica c	P. inefficax d	P. shirazica e	P. parafulva DSM 17004 T	P. fulva IAM 1529 T	Pseudomonas sp. W15Oct28	P. putida ATCC T 12633	P. 700476 monteilii ATCC	P. alloputida f
	Flagellation	multipl e polar	single polar	single polar	single polar	two polar	single polar	single polar	single polar	single polar	single polar	2 to 3 polar	single polar	ND	single polar
	GEN III MicroPlate (28°C)
	6% NaCl	-	+	+	+	ND	-	-	-	+	+	-	-	+	-
	Acetic acid	+	+	+	+	-	+	+	+	+	+	-	+	+	+
	Bromo-succinic acid	+

Table 3

 3 Description of Pseudomonas alloputida sp. nov. according to Digital Protologue TA00611 assigned by the www.imedea.uib.es/dprotologue website

	Taxonumber	TA00611
	Species name	Pseudomonas alloputida
	Genus name	Pseudomonas
	Specific epithet	alloputida
	Species status	sp. nov.
	Species etymology	al.lo.pu'ti.da. Gr. masc. adj. allos, other; L. fem. adj. putida, rotten,
		stinking; specific epithet of a Pseudomonas; N.L. fem.
		adj. alloputida, another putida.
	Authors	Vahid Keshavarz-Tohid, Jordan Vacheron, Audrey Dubost, Claire
		Prigent-Combaret, Parissa Taheri, Saeed Tarighi, Seyed Mohsen
		Taghavi, Yvan Moënne-Loccoz, Daniel Muller

Title

Genomic, phylogenetic and catabolic re-assessment of the Pseudomonas putida clade supports the delineation of Pseudomonas alloputida sp. nov., Pseudomonas inefficax sp. nov., Pseudomonas persica sp. nov., and Pseudomonas shirazica sp. nov.

2 conditions for strain testing Aerobe

		Colonies were fluorescent, approximately 3 mm in diameter,
		circular and convex with an entire margin
	Lowest temperature for growth	15
	Highest temperature for growth	30
	Temperature optimum	28
	pH optimum	7.5
	pH category	Neutrophile
	Lowest NaCl concentration for growth	1%
	Highest NaCl concentration for growth	8%
	Relationship to O 2	Halotolerant (optimum < 1 % NaCl and growth observed at > 6 %
		NaCl)
	O

guariconensis LMG27394 T Pseudomonas fulva CIP106765 T Pseudomonas parafulva DSM117004 T Pseudomonas putida NBRC 14164 T

  84.3 83.5 83.5 83.6 84.1 100 86.5 87.1 87.0 86.2 86.8 86.9 82.1 83.8 84.9 87.3 87.6 86.6 86.6 86.7 87.2 87.7 87.9 87.8 87.8 88.0 83.3 82.8 82.9 83.0 83.2 86.4 100 85.7 85.7 84.9 85.5 85.6 80.8 83.3 84.1 85.8 86.1 85.5 85.5 85.5 86.0 86.5 86.4 86.4 86.4 86.6 Pseudomonas mosselii BW11M1 84.8 84.7 83.7 83.8 84.1 84.5 87.1 85.8 100 99.2 90.7 91.3 89.6 82.6 83.6 85.3 86.2 86.4 85.7 85.7 85.8 86.2 86.8 86.9 86.7 86.7 86.9 84.7 83.7 83.7 83.9 84.5 87.0 85.7 99.2 100 90.7 91.2 89.6 82.7 83.6 85.3 86.3 86.3 85.8 85.9 86.0 86.8 86.9 86.9 86.9 86.6 87.0 83.6 82.9 82.9 83.2 83.4 86.2 84.9 90.9 90.8 100 94.7 88.3 70.0 83.0 84.6 85.3 85.5 84.7 84.7 84.8 85.2 85.9 86.2 86.1 86.1 86.3 Pseudomonas soli RUB1 84.4 84.5 83.6 83.7 83.9 84.3 86.9 85.5 91.3 91.3 94.9 100 89.1 82.4 83.4 85.0 86.3 86.3 85.5 85.5 85.7 86.1 86.5 86.7 86.6 86.5 86.8 84.4 83.6 83.6 83.8 84.3 86.8 85.6 89.6 89.5 88.4 89.1 100 82.7 83.4 85.2 86.2 86.3 85.7 85.7 85.8 86.2 86.8 86.8 86.8 86.7 87.0 80.9 79.9 80.1 79.7 80.0 81.9 81.1 82.9 82.6 70.0 82.4 82.7 100 78.4 80.5 81.5 81.6 81.4 81.4 82.7 81.6 82.3 82.4 82.5 82.4 83.2 81.9 81.5 81.5 82.3 82.5 84.0 83.4 83.6 83.5 83.1 83.4 83.4 79.0 100 82.8 84.3 84.4 83.9 84.0 84.0 84.3 84.4 84.5 84.5 84.5 84.6 83.2 82.7 82.9 83.5 83.8 84.9 84.1 85.5 85.4 84.4 85.2 85.4 80.7 82.8 100 84.3 84.4 84.0 84.1 84.0 84.2 84.9 84.8 84.5 84.5 84.8 83.9 83.0 83.2 83.2 83.8 87.2 85.8 86.1 86.3 85.2 86.2 86.1 81.6 84.2 84.3 100 94.7 90.2 90.1 90.2 90.5 90.1 90.2 90.0 89.9 90.2 Pseudomonas sp. W15Oct28 83.9 83.9 83.4 83.5 83.2 83.8 87.4 86.0 86.3 86.2 85.4 86.3 86.3 81.8 84.3 84.3 94.7 100 90.1 90.1 90.1 90.7 90.2 90.4 90.2 90.1 90.6 Pseudomonas alloputida VKh14 83.5 83.4 70.0 82.8 82.6 83.3 86.6 85.5 85.7 85.9 84.4 85.5 85.7 81.5 83.8 83.8 90.2 90.2 100 100 97.1 89.5 89.5 89.6 89.5 89.4 89.7

				Pseudomonas sp. JV241A	Pseudomonas donghuensis HYS T	P. vranovensis DSM 16006 T	Pseudomonas alkylphenolia KL28 T	Pseudomonas sp. USDA-ARS-USMARC-56711	P. cremoricolorata DSM 17059 T	P. plecoglossicida NBRC 103162 T	P. taiwanensis DSM 21245 T	Pseudomonas mosselii BW11M1	Pseudomonas mosselii ATCCBAA 99 T	Pseudomonas soli LMG27941 T	Pseudomonas soli RUB1	Pseudomonas entomophila L48	Pseudomonas sp. W15Oct28	Pseudomonas alloputida VKh14	Pseudomonas alloputida VKh7 T	Pseudomonas alloputida KT2440	T Pseudomonas monteilii DSM14164	Pseudomonas monteilii SB3078	Pseudomonas shirazica VM14 T	T Pseudomonas inefficax JV551A3	Pseudomonas inefficax JV551A1	Pseudomonas persica RUB6 T
	Pseudomonas sp. JV241A			100 93.3 86.9 87.4 82.8 83.2 84.3 83.3 84.7 84.7 83.5 84.5 84.6 81.2 81.9 83.1 84.0 84.1 83.4 83.4 83.5 83.9 84.3 84.2 84.2 84.1 84.5
	Pseudomonas donghuensis HYS	T		93.4 100 87.0 87.2 82.8 83.1 84.4 83.4 84.7 84.7 83.6 84.4 84.5 81.0 81.9 83.2 84.0 84.0 83.4 83.4 83.4 83.8 84.3 84.3 84.0 84.1 84.4
	Pseudomonas vranovensis DSM 16006 T	87.0 86.8 100 87.3 82.1 82.4 83.5 82.7 83.7 83.7 82.9 83.5 83.6 79.8 81.5 82.5 83.1 83.4 82.7 82.7 82.8 83.2 83.3 83.4 83.2 83.3 83.5
	Pseudomonas alkylphenolia KL28	T	87.5 87.3 87.3 100 82.2 82.5 83.6 82.8 83.8 83.7 82.6 83.7 83.7 80.2 81.6 82.7 83.2 83.5 82.7 82.8 82.9 83.1 83.4 83.3 83.4 83.3 83.6
	Pseudomonas sp. USDA-ARS-USMARC-56711	83.0 82.7 82.1 82.2 100 83.3 83.7 82.8 84.2 84.1 83.2 83.8 84.0 79.6 82.3 83.4 83.3 83.3 82.8 82.8 82.9 83.3 83.6 83.7 83.5 83.5 83.9
	Pseudomonas cremoricolorata DSM 17059 T	83.4 83.2 82.4 82.5 83.3 100 84.4 83.4 84.5 84.5 83.4 84.4 84.3 80.3 82.5 83.8 83.8 83.9 83.5 83.6 83.4 83.7 84.1 84.3 84.0 84.0 84.4
	Pseudomonas plecoglossicida NBRC 103162
	Pseudomonas taiwanensis DSM 21245
	Pseudomonas mosselii ATCCBAA 99
	Pseudomonas soli LMG27941
	Pseudomonas entomophila L48
	Pseudomonas guariconensis LMG27394
	Pseudomonas fulva CIP106765
	Pseudomonas parafulva DSM117004
	Pseudomonas putida NBRC 14164
																pyruvate, p-hydroxy-
						phenylacetic acid, propionic acid, quinic acid, γ-amino butyric acid, β-
						hydroxybutyric, formic acid, aztreonam, vancomycin, guanidine HCl,
						lincomycin, minocycline, niaproof 4, rifamycin SV, tetrazolium blue,
						tetrazolium violet, troleandomycin, 1% NaCl, 1% sodium lactate, 4% NaCl,
						D-serine, D-fucose, fusidic acid, L-arginine, L-galactonic acid lactone, L-
						malic acid, lithium chloride, mucic acid, nalidixic acid, pH 5, pH 6, sodium
						bromate, sodium butyrate
	Variable tests with BIOLOG					Tween 40, α-keto butyric acid
	Energy metabolism					Chemoorganotroph

T P. T 84.3 T 83.3 T 84.6 T 83.7 T 84.4 T 81.3 T 82.0 T 83.3 T 83.9

Pseudomonas alloputida VKh7 T 83

  .4 83.4 70.0 82.8 82.6 83.3 86.7 85.5 85.7 85.8 84.6 85.5 85.6 81.2 83.9 83.8 90.3 90.2 100 100 97.1 89.5 89.5 89.7 89.5 89.4 89.8 Pseudomonas alloputida KT2440 83.5 83.5 82.9 83.0 82.9 83.4 86.7 85.5 85.8 86.1 85.0 85.7 85.9 82.7 83.9 84.0 90.3 90.2 97.1 97.1 100 89.7 89.8 89.7 89.6 89.6 89.8

Pseudomonas monteilii DSM14164 T 83

  .9 83.9 83.2 83.1 83.2 83.6 87.2 85.9 86.3 86.8 85.2 86.1 86.3 81.3 84.4 84.2 90.6 90.9 89.6 89.6 89.7 100 90.0 90.2 90.0 89.9 90.5 Pseudomonas monteilii SB3078 84.3 84.2 83.3 83.4 83.4 83.9 87.7 86.5 86.9 86.9 86.1 86.6 86.8 82.5 84.3 84.7 90.2 90.3 89.5 89.5 89.8 90.0 100 97.5 94.4 94.4 93.7 84.3 84.2 83.4 83.4 83.5 84.1 87.8 86.4 86.9 86.9 86.1 86.8 86.8 82.1 84.4 84.6 90.2 90.4 89.6 89.6 89.7 90.1 97.5 100 94.7 94.6 93.8 Pseudomonas inefficax JV551A3 T 84.2 84.0 83.3 83.4 83.5 84.0 87.8 86.3 86.8 87.0 86.0 86.5 86.8 82.1 84.4 84.5 90.0 90.2 89.6 89.5 89.6 90.0 94.3 94.5 100 99.9 93.6 Pseudomonas inefficax JV551A1 84.3 84.1 83.2 83.4 83.4 84.0 87.7 86.3 86.7 86.6 85.9 86.5 86.7 81.9 84.3 84.4 89.9 90.1 89.4 89.4 89.5 89.9 94.3 94.6 99.9 100 93.5 84.5 84.5 83.6 83.5 83.7 84.2 87.9 86.5 87.0 87.0 86.1 86.9 87.0 83.0 84.6 84.8 90.4 90.7 89.8 89.7 89.9 90.3 93.8 93.8 93.7 93.7 100

	Pseudomonas shirazica VM14
	Pseudomonas persica RUB6

T T

Acknowledgments 12 This research was supported by Academic Research cluster 3 of Région ne-Alpes, and CNRS (France). We thank the Vice-Chancellor's office for Research and Technology in Agricultural Science and Environmental Research of Khuzestan, Iran, for their support. We thank Danis Abrouk (iBio), Florence Gerin and Laurence Loiseau (PARMIC) from UMR Ecologie Microbienne for technical help and discussion. We are very grateful to Jian Woon and Chan Kok Gan (University of Malaya) for strain DSM 17059 T , Maarten Ghequire (Catholic University Leuven) for strain BW11M1, Sandra Matthijs and Pierre Cornelis (Université Libre de Bruxelles) for strain W15Oct28, Morten Simonsen Dueholm (Aalborg University) for strain SB3078. We also acknowledge the LABGeM and the National Infrastructure France Génomique for support within the MicroScope annotation platform.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Submitter

Daniel MULLER E-mail of the submitter daniel.muller@univ-lyon1.fr

Designation of the type strain RUB6

Strain collection numbers CFBP 8486=LMG 29757

16S rRNA gene accession number LT718462

Genome accession number [EMBL] PRJEB25066

Genome status Draft

Genome size 5425242

GC mol % 62.92

Country of origin Iran

Region of origin

Submitter

Daniel MULLER E-mail of the submitter daniel.muller@univ-lyon1.fr

Designation of the type strain VM14

Strain collection numbers CFBP 8487 = LMG 29953

16S rRNA gene accession number LT718474

Genome accession number [EMBL] PRJEB25068

Genome status Draft

Genome size 5,514,185 GC mol % 62.84

Country of origin Iran

Region of origin Mazandaran province

Date of isolation

Number of strains in study 5

Source of isolation of non-type strains

Bean rhizosphere

Growth medium, incubation conditions

King's B agar (KBA) at 28°C

Gram stain Negative

Cell shape Rod

Motility Motile

Submitter

Daniel MULLER E-mail of the submitter daniel.muller@univ-lyon1.fr

Designation of the type strain JV551A3

Strain collection numbers CFBP8493 = DSM 108619

16S rRNA gene accession number PRJEB24815

Genome accession number [embl] PRJEB24815

Genome status Draft

Genome size 6240036

GC mol % 62.85

Country of origin France

Region of origin

Béligneux "AIN 01" Pseudomonas strains with sequenced taxonomic markers (blue dots). Cellvibrio japonicus Uedea 107 T was used as outgroup. In the figure, the groups and phylogenetic lineages proposed previously [START_REF] Peix | The current status on the taxonomy of Pseudomonas revisited: An update[END_REF][START_REF] Mulet | DNA sequence-based analysis of the Pseudomonas species[END_REF][START_REF] Hesse | Genome-based evolutionary history of Pseudomonas spp[END_REF] are indicated; type strains and proposed type strains are in bold. Within the Pseudomonas putida group, the Pseudomonas putida clusters proposed previously [START_REF] Keshavarz-Tohid | Phylogenetic diversity and antagonistic traits of root and rhizosphere pseudomonads of bean from Iran for controlling Rhizoctonia solani[END_REF] are highlighted in blue rectangles; the dotted lines are delimiting clusters of strains belonging to the same species according to ANI data. Concatenated sequences were used to compute the tree using PhyML [START_REF] Guindon | New algorithms and methods to estimate Maximum-Likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF], with 500 bootstraps, and SeaView v4 [START_REF] Gouy | SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF]. The tree was visualized using iTOL software [START_REF] Letunic | Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees[END_REF]

Date of isolation

Supplementary tables

Added in an Excel file:

Table S1. Genome accession numbers.

Table S2. rrs, rpoD and gyrB accession numbers for strains without available genome sequence.

Table S3. Genomic relationship between strains in the Pseudomonas genus based on ANI values (%), which were determined with fastANI [START_REF] Jain | High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[END_REF]. Type strains are in bold and the four new species in red. Blue strains are not presented in the main figures or tables.

Table S4. Digital DNA-DNA hybridization (dDDH) estimates computed using Genome-to-Genome Distance Calculator GGDC 2.1 [START_REF] Meier-Kolthoff | Genome sequence-based species delimitation with confidence intervals and improved distance functions[END_REF][START_REF] Meier-Kolthoff | Complete genome sequence of DSM 30083T, the type strain (U5/41T) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy[END_REF][START_REF] Meier-Kolthoff | Taxonomic use of DNA G+C content and DNA-DNA hybridization in the genomic age[END_REF]. Strains are listed according to the new species proposed.

Table S5. Differential phenotypic characteristics of strains of the P. putida group. Data are shown for the type strain as well as other related strains of the same species. Reference is cited for tests found in the literature. When no references are indicated data were obtained in present work. In yellow the mean phenotype of the species.

Table S6. Genome size and GC% content of strains in the P. putida group.

Table S7. Features of genomes used in the study.