# Collimonas rhizosphaerae sp. nov., a novel species isolated from the beech rhizosphere

Stéphane Uroz, Ségolène Bouche, Emmanuelle Morin, Mathilde Bocquart, Ravi Kumar, Michael W Rey, Jonathan Pham, Fidel Akum, Johan H J Leveau

► To cite this version:

Stéphane Uroz, Ségolène Bouche, Emmanuelle Morin, Mathilde Bocquart, Ravi Kumar, et al. *Collimonas rhizosphaerae* sp. nov., a novel species isolated from the beech rhizosphere. *International Journal of Systematic and Evolutionary Microbiology*, 2024, 74 (7), pp.006481. 10.1099/ijsem.0.006481 hal-04672474

HAL Id: hal-04672474

https://hal.science/hal-04672474v1

Submitted on 19 Aug 2024

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# *Collimonas rhizosphaerae* sp. nov., a novel species isolated from the beech rhizosphere

Stephane Uroz¹,²*, Ségolène Bouche¹,², Emmanuelle Morin¹, Mathilde Bocquart¹, Ravi Kumar³, Michael W. Rey³, Jonathan Pham³, Fidel Akum⁴ and Johan H. J. Leveau⁴

## Abstract

Bacterial strain H4R21T was isolated from beech rhizosphere soil sampled in the forest experimental site of Montiers (Meuse, France). It effectively weathers minerals, hydrolyses chitin and produces quorum sensing signal molecules. The strain is aerobic and Gram-stain-negative. Phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain H4R21T belongs to the genus *Collimonas* with high sequence similarity to *C. arenae* Ter10T (99.38%), *C. fungivorans* Ter6T (98.97%), *C. pratensis* Ter91T (98.76%), *C. humicola* RLT1W51T (98.46%) and *C. silvisoli* RXD178T (98.46%), but less than 98% similarity to other strains of the genus *Collimonas*. The predominant quinone in H4R21T is ubiquinone-8 (Q8). The major polar lipids are diphosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and lipid. The major fatty acids identified were C₁₂:₀, C₁₂:₀ 3-OH, C₁₆:₀ and C₁₇:₀ cyclo. The digital DNA G+C content of the genomic DNA was 59.5mol%. Furthermore, the strain could be clearly distinguished from its closely related type strains by a combination of phylogenomic and *in silico* DNA-DNA hybridization results, and phenotypic characteristics. Therefore, strain H4R21T represents a novel species within the genus *Collimonas*, for which the name *Collimonas rhizosphaerae* sp. nov. is proposed, with strain H4R21T (=CFBP 9203T=DSM 117599T) as the type strain.

## INTRODUCTION

The genus *Collimonas* belongs to the family *Oxalobacteraceae* and is considered as a member of the rare biosphere, since metagenomic analyses reveal that its abundance in soil compartments is typically very low [1-5]. Paradoxically, though rarely found in metagenomic studies, *Collimonas* strains are culturable and can be isolated relatively easily [5-7]. In term of ecology, representatives of the genus *Collimonas* are typically found in nutrient-poor and acidic soils with limited human disturbance and usually rich in fungi, suggesting an adaptation to oligotrophic conditions [4, 8-12]. Microcosm experiments have validated this hypothesis, showing that even a low input of potassium or magnesium in a soil that is naturally poor in these nutrients induced a fast reduction of the abundance of *Collimonas* [7]. Members of the genus *Collimonas* are Gram-stain negative, aerobic rods and usually motile, although with some exceptions [9-14]. Regarding the functional potential of *Collimonas*, independent screenings of different collections have revealed a conserved ability to hydrolyse chitin and to utilize fungal hyphae as carbon source, to inhibit fungal growth, to weather minerals, and to produce quorum-sensing signal molecules [8, 15-21]. The ability to inhibit fungal growth was shown to depend on various molecules (e.g. collimonin and carenaemin) [18, 19]. Noticeably, some *Collimonas* have been shown to promote plant growth [20]. *Collimonas* represent an interesting genus with promising properties, which invites deeper analyses into their taxonomy and functional abilities. Currently, six *Collimonas* species names have been validly published, namely *C. anthrihumi*, *C. arenae*, *C. fungivorans*, *C. pratensis*, *C. humicola* and *C. silvisoli* [9-12].

**Author affiliations:** ¹Université de Lorraine, INRAE, UMR1136, Interactions Arbres-Microorganismes, 5400 Nancy, France; ²INRAE, UR1138, Biogéochimie des Ecosystèmes Forestiers, F-54280 Champenoux, France; ³Novozymes Inc., 1445 Drew Ave., Davis, CA 95618, USA; ⁴Department of Plant Pathology, University of California, Davis, CA 95616, USA.

*Correspondence: Stephane Uroz, stephane.uroz@inrae.fr

Keywords: beech forest soil; *Collimonas rhizosphaerae*; *Oxalobacteraceae*.

Abbreviations: ANI, average nucleotide identity; dDDH, digital DNA-DNA hybridization.

Whole-genome and raw sequences: GCA_038896935.1 and JBANDC010000000. 16S rRNA gene sequence: PP833028.

A supplementary figure is available with the online version of this article.

006481 © 2024 The Authors

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In this study, we performed a polyphasic taxonomic and functional characterization of strain H4R21T. Our analyses provide phenotypic, genomic and phylogenomic evidence that this strain meets the criteria for delineating a new species of *Collimonas*. Strain H4R21T is of special interest as it is able to metabolize fungal derivates (e.g., chitin, trehalose, mannitol), to solubilize inorganic phosphorus and complex minerals (e.g., biotite, garnet, apatite) [22], to inhibit fungal growth and to produce quorum-sensing signal molecules.

## ISOLATION AND ECOLOGY

Strain H4R21T is part of a collection of 370 bacterial strains that was established during a study on the functional abilities of bacterial isolates from beech rhizosphere and their comparison with the properties of bacteria from bulk soil [15]. Sampling was done on the forest experimental site of Montiers (48.53 N, 5.32E) which is located in the Meuse department (in northeastern France) under mono-specific plots of beech trees. After removal of the litter layer, cores (33×33 cm, and 5–20 cm deep, representing the organo-mineral soil horizon) were collected. From the rhizosphere soil of one of these cores, strain H4R21T was isolated by serial dilutions on 1/10 diluted tryptic soy agar (TSA) medium [Difco's tryptic soy broth (TSB) 3 g l⁻¹ and agar 15 g l⁻¹] with a pH adjusted to 6.5. The isolate was purified by three successive spreads on the same medium to obtain a pure culture. Strain H4R21T was grown routinely on 1/10 TSA (or 1/10 TSB) or on low salt LB (LBm) medium for 3 days at 25 °C, under agitation (150 r.p.m.) for liquid media. Its purity was routinely confirmed by macro- and microscopic observations, by chitin degradation assay (description below) and by sequencing of its 16S rRNA gene and its genome. Stock cultures were stored at –80 °C in LBm medium plus 40% glycerol. Chemical characteristics of the soil samples from which strain H4R21T was isolated were as follows: pH 4.63±0.04, 17.20±0.74 g kg⁻¹ total carbon, 1.19±0.07 g kg⁻¹ total nitrogen, 0.38±0.13 g kg⁻¹ phosphorus extracted according to the Duchaufour method and 29.78±13.92 g kg⁻¹ organic matter [15].

Strain H4R21T (DSM 117599T = CFBP 9203T) is available in the following public culture collections: Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; www.dsmz.de/collection) and the CIRM-CFBP (Collection française des bactéries associées aux plantes). Reference strains *C. humicola* RLT1W51T (KACC 21985T), *C. silvisoli* RXD178T (KACC 21987T), *C. antrihumi* C3-17T (DSM 104040T), *C. arenae* Ter10T (DSM 21398T), *C. pratensis* Ter91T (DSM 21399T), *C. fungivorans* Ter 6T (DSM 17622T) and Ter331, as well as *C. arenae* Ter282, *Collimonas* sp. Cal35 and *C. pratensis* PMB3(1) were obtained from lab collections, the DSMZ or from the Korean Agricultural Culture Collection (KACC) and included in our analyses for comparison to H4R21T.

## 16S rRNA GENE PHYLOGENY

Genomic DNA of strain H4R21T was extracted according to Pospiech and Neumann [23] after a sequential treatment with: (i) lysozyme (1 mg ml⁻¹) at 37 °C for 1 h, (ii) addition of 1/10 of sodium dodecyl sulphate (final concentration, 1%) and (iii) proteinase K (final concentration, 1 mg ml⁻¹). DNA was recovered after chloroform purification and isopropanol precipitation. Full-length 16S rRNA gene sequences were recovered from isolate H4R21T and all reference strains, and aligned in Seaview with MUSCLE [24]. A phylogenetic tree was reconstructed using the PhyML algorithm with *Caballeronia mineralivorans* PML1(12)T as outgroup.

The 16S rRNA gene sequence comparisons and phylogenetic tree analyses confirmed that strain H4R21T is a member of the genus *Collimonas*. Comparative analysis of the 16S rRNA gene sequence of strain H4R21T (accession number: PP833028) with those of other *Collimonas* type species revealed that this strain is closely related to species *C. areneae* Ter10T (99.38%), *C. fungivorans* Ter6T (98.97%), *C. pratensis* Ter91T (98.76%), *C. humicola* RLT1W51T and *C. silvisoli* RXD178T (both 98.46%), and had less than 98% similarity to other members of the genus *Collimonas*. However, the phylogenetic tree revealed that this new strain forms a phylogenetic branch that is separated from all the other published type strains of the genus, as suggested by the bootstrap value of 92% (Fig. 1). Strain H4R21T is also separated from the other *Collimonas*-related genera (*Herbaspirillum*, *Janthinobacterium*, *Massilia*, *Herminiimonas*).

## CORE GENE-BASED PHYLOGENOMIC ANALYSES

The whole genome of strain H4R21T was sequenced as 2×150 bp paired reads on an Illumina NextSeq 500 instrument (accession numbers of whole-genome sequence and raw sequences: GCA_038896935.1 and JBANDC010000000). The assembly was generated with SPAdes version 3.9.0 [25]. The draft genome is 5.6 Mb and is composed of a total of 46 contigs larger than 500 bp. Similar genome sizes have been reported for other species of *Collimonas* (5.7 Mb for *C. pratensis* Ter91T, 5.6 Mb for *C. fungivorans* Ter6T), while a smaller genome size of 4.7 Mb was observed for *C. arenae* Ter10T (and Ter282) and *C. antrihumi* C3-17T (Table 1) [11–14]. No plasmid feature was detected, as was the case in the genomes of *C. fungivorans* Ter331 and *C. arenae* Cal35. The genome of strain H4R21T has a G+C content of 59.52mol%, similar to the G+C contents reported for *C. silvisoli* RXD178T (59.45mol%) and *C. humicola* RLT1W51T (59.4mol%), but higher than other species (54.6mol% for *C. antrihumi* C3-17T and 58.99mol% for *C. fungivorans* Ter6T) (Table 1) [11–14].

The assembled genomes of other *Collimonas* strains were obtained from the GenBank database for comparison to the genome of strain H4R21T. TYGS [26] was used to generate the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) relatedness based on genome sequences. BUSCO analysis (version 5.6.1 [27]) showed a genome completeness at 99.9% as compared to the *Burkholderiales* odb10 lineage data set. According to the BUSCO marker-based phylogenetic tree that was inferred using 527
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Maximum-likelihood (PhyML) 16S rRNA gene phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain H4R21T and other closely related genera and species in the family *Oxalobacteraceae*. Bootstrap values are expressed as a percentage of 1000 replications. Only branch-support values ≥50% are included.</figcaption></figure>

single-copy genes with RAxML-NG (version 0.9.0) [28] applying a partitioned analysis and 1000 bootstraps replicates, strain H4R21T clustered closest with *C. fungivorans* Ter6T, *C. antrihumi* C3-17T and *C. silvisoli* RXD178T (Fig. 2). ANI values between strain H4R21T and the closely related type species were calculated as ranging between 83.79 and 90.82%. Correspondingly, the dDDH values ranged from 35.5% with *C. arenea* Cal35 to 60.2% with *C. fungivorans* Ter6T(Table 1). Considering the recognized threshold values (ANI 95-96% and dDDH 70%), strain H4R21T represents a novel *Collimonas* species.

## MORPHOLOGY AND PHYSIOLOGY

Cell morphology and motility were observed with a BX41 Olympus microscope at a magnification of ×1000 and by using phase contrast and differential interference contrast with unstained living bacteria. A Gram characterization was performed using the aminopeptidase test from Sigma. For all the assays described below, the bacterial inoculum was prepared as follows. Bacterial strains were grown for 2 days in liquid LBm, centrifuged at 7000 g and washed twice with sterile milliQ water. The optical density
<caption>**Table 1.** Differential biochemical characteristics of all examined strains of species of the genus *Collimonas*</caption>

Strains: 1, *C. rhizosphaerae* H4R21T; 2, *C. antrihumi* C3-17T; 3, *C. humicola* RLT1W51T; 4, *C. silvisoli* RXD178T; 5, *C. arenae* Ter10T; 6, *C. arenae* Ter282; 7, *C. arenae* Cal35; 8, *C. fungivorans* Ter6T; 9, *C. pratensis* Ter91T; 10, *C. pratensis* PMB3(1). Differential biochemical characteristics of all examined strains of species of the genus. +, Positive; –, negative; w, positive with a weak signal. All Biolog, chitin hydrolysis and iron chelating data were generated in this study. The data presented for temperature, pH and NaCl ranges of the reference bacterial strains come from the literature.

<table><thead><tr><th>Characteristics</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th></tr></thead><tbody><tr><td><strong>Genomic features</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Size (Mb)</td><td>5.6</td><td>4.72</td><td>5.63</td><td>5.66</td><td>4.7</td><td>4.7</td><td>5.6</td><td>5.6</td><td>5.7</td><td>5.6</td></tr><tr><td>G+C content (mol%)</td><td>59.52</td><td>54.6</td><td>59.4</td><td>59.45</td><td>57</td><td>57</td><td>56</td><td>58.99</td><td>58.5</td><td>59</td></tr><tr><td><strong>Physiological and functional features</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Temperature range for growth (15-30 °C)</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>pH range for growth (pH 5-8)</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>NaCl range for growth (0.5-2%)</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Chitin hydrolysis</td><td>+</td><td>-</td><td>+</td><td>w</td><td>w</td><td>w</td><td>+</td><td>+</td><td>w</td><td>+</td></tr><tr><td>Chelating activity</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td><strong>Utiliation of substrate (Biolog)</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>2-Aminoethanol</td><td>+</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>α-Cyclodextrin</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>α-D-Glycerol-1-phosphate</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>α-D-Lactose</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>α-Hydroxybutyric acid</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>α-Ketobutyric acid</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>α-Ketovaleric acid</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Acetic acid</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>Adonitol</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>Methyl β-D-glucoside</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Bromosuccinic acid</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td></tr><tr><td>D-Galacturonic acid</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>D-Gluconic acid</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D-Glucosaminic acid</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D-Glucose-6-phosphate</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td>D-Psicose</td><td>+</td><td>-</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>D-Raffinose</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>D-Sorbitol</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Trehalose</td><td>+</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D-Alanine</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D-Arabitol</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Cellobiose</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D-Galactonic acid lactone</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D-Mannitol</td><td>+</td><td>-</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D-Mannose</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr></tbody></table>

Continued
<table><caption>**Table 1. Continued**</caption><thead><tr><th>Characteristics</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th></tr></thead><tbody><tr><td>Melibiose</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D-Serine</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D,L-α-Glycerol phosphate</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>D,L-Carnitine</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Dextrine</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>γ-Hydroxybutyric acid</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td></tr><tr><td>Gentibiose</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Glucuronamide</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Glycerol</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Glycogen</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Hydroxy-L-proline</td><td>-</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td>I-Erythritol</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>Inosine</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>L-Alanine</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td></tr><tr><td>L-Histidine</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td></tr><tr><td>L-Leucine</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>L-Ornithine</td><td>+</td><td>-</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>L-Phenylalanine</td><td>-</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>L-Rhamnose</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>L-Threonine</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td></tr><tr><td>Lactulose</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>Malonic acid</td><td>+</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>Maltose</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>N-Acetyl-D-galactosamine</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>p-Hydroxyphenylacetic acid</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Phenylethylamine</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>Quinic acid</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td></tr><tr><td>Succinic acid monomethyl ester</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Sucrose</td><td>-</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Turanose</td><td>-</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Uridine</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Urocanic acid</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Xylitol</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td></tr></tbody></table>

All the following substrates are metabolized by all the *Collimonas* strains tested: α-D-glucose, α-ketoglutaric acid, β-hydroxybutyric acid, cis-aconitic acid, citric acid, D-glucoronic acid, D-fructose, D-galactose, D-saccharic acid, D,L-lactic acid, formic acid, γ-aminobutyric acid, glycyl-L-aspartic acid, glycyl-L-glutamic acid, L-alainamide, L-allanyl-glycine, L-arabinose, L-asparagine, L-aspartic acid, L-fucose, L-glutamic acid, L-proline, L-pyroglutamic, L-serine, m-inositol, N-acetyl-D-glucosamine, propionic acid, pyruvic acid methyl ester, succinamic acid, succinic acid, Tween 40 and Tween 80. All the following substrates are not metabolized by all the *Collimonas* strains tested: 2,3 butanediol, itaconic acid, putrescine, sebacic acid and thymidine.
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** BUSCO marker-based phylogenetic tree showing the relationship between strain H4R21T and other closely related genera and species in the family Oxalobacteraceae. The phylogenetic tree was inferred using 527 single-copy genes. Bootstrap values are expressed as a percentage of 1000 replications.</figcaption></figure>

(OD=595 nm) of each bacterial suspension was adjusted to 0.9 (corresponding to ca. $1 \times 10^8$ c.f.u. ml$^{-1}$). Antibiotic susceptibility was tested in liquid LBm with ampicillin, chloramphenicol, kanamycin, streptomycin (all at final concentration: 100, 150 and 200 µg ml$^{-1}$), gentamicin (final concentration: 20 and 50 µg ml$^{-1}$) and tetracycline (final concentration: 10, 20 and 50 µg ml$^{-1}$). Motility was tested on M9 mineral medium (20 mM NH$_4$Cl; 12 mM Na$_2$HPO$_4$; 22 mM KH$_2$PO$_4$; 8.6 mM NaCl; 1 mM MgSO$_4$; 1 mM CaCl$_2$; 0.2% glucose; and 0.5% casamino acids) amended with 0.25% agar, following the recommendation of Turnbull
and Whitchurch [29]. A volume of 1 µl bacterial inoculum was spotted on the centre of the agar in the Petri dish and colony diameters were scored after 2 days. Growth at different temperatures (15, 20, 25, 30 and 35 °C) was assessed in LBm medium after incubation for 10 days. The pH range for growth (pH 3.0–10.0, at intervals of 1.0 pH unit) was also tested in LBm medium, as was tolerance of increasing NaCl concentrations (0.5–6%, with an interval of 1%, w/v). For all these assays, growth was determined by measuring OD at 595 nm with a spectrophotometer (Bio-Rad, iMark). The ability to hydrolyse chitin was tested on minimal agar medium containing colloidal chitin (per litre: 5 g NaCl, 1 g KH₂PO₄, 0.1 g yeast extract, 20 g agar, and 2 g colloidal chitin) [21]. After incubation at 25 °C for 14 days, the clearing of the initially turbid medium indicated chitin hydrolysis and the diameters of the resulting halos were measured. The production of N-acyl homoserine lactone (AHL) was assessed in T-streak assays [30] with the *Chromobacterium violaceum* CV026 biosensor [31]. The production of violacein was scored after 2 and 4 days of incubation. The siderophore activity was performed according to Schwyn and Neilands [32], after a 2 day incubation in liquid AB medium (devoid of iron) amended with citrate as sole carbon source (2 g l⁻¹), by mixing a 1:1 volume of supernatant and a volume of chromazurol S (CAS) solution. After 1 h of incubation at 20 °C, the OD was measured at 655 nm with a spectrophotometer (Bio-Rad, iMark). The ability to utilize different carbon substrates was determined for each bacterial strain using the GN2 microplates (Biolog), which permits simultaneous testing of 95 substrates. OD values were recorded at 595 nm and considered positive ‘+’ for OD values greater than 0.1 and negative ‘0’ for OD values lower than 0.1.

Cells of strain H4R21T are non-spore-forming and shaped rods (approx. 2–3 µm long and 1 µm wide). The different assays performed revealed that strain H4R21T is aerobic, Gram-negative and motile, and susceptible to chloramphenicol, gentamicin, kanamycin, streptomycin and tetracycline, but resistant to ampicillin (up to 200 µg ml⁻¹). The observation of motility was consistent with the presence of genes encoding flagellar biosynthesis and assembly as well as the type IV pili system in the genome of H4R21T. Noticeably, flagellar biosynthesis and assembly encoding genes were detected in the genomes of the motile strains of

<table><caption>**Table 2.** Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) relatedness (%) based on genome sequences</caption><thead><tr><th colspan="11">ANI score</th></tr><tr><th>Strain</th><th>Cal35</th><th>C3-17<sup>T</sup></th><th>H4R21<sup>T</sup></th><th>PMB3(1)</th><th>RLT1W51<sup>T</sup></th><th>RXD178<sup>T</sup></th><th>Ter10<sup>T</sup></th><th>Ter282</th><th>Ter6<sup>T</sup></th><th>Ter91<sup>T</sup></th></tr></thead><tbody><tr><td>Cal35</td><td>100</td><td>83.34</td><td>83.80</td><td>85.16</td><td>85.49</td><td>83.48</td><td>82.63</td><td>82.63</td><td>83.73</td><td>85.05</td></tr><tr><td>C3-17<sup>T</sup></td><td>83.31</td><td>100</td><td>85.89</td><td>83.87</td><td>84.20</td><td>87.05</td><td>83.17</td><td>83.21</td><td>85.77</td><td>83.86</td></tr><tr><td><strong>H4R21<sup>T</sup></strong></td><td><strong>83.79</strong></td><td><strong>85.93</strong></td><td><strong>100</strong></td><td><strong>85.86</strong></td><td><strong>86.23</strong></td><td><strong>86.94</strong></td><td><strong>83.83</strong></td><td><strong>83.88</strong></td><td><strong>90.82</strong></td><td><strong>85.79</strong></td></tr><tr><td>PMB3(1)</td><td>85.15</td><td>83.94</td><td>85.91</td><td>100</td><td>90.22</td><td>84.36</td><td>83.73</td><td>83.79</td><td>86.33</td><td>98.17</td></tr><tr><td>RLT1W51<sup>T</sup></td><td>85.39</td><td>84.26</td><td>86.29</td><td>90.11</td><td>100</td><td>84.81</td><td>83.84</td><td>83.85</td><td>86.45</td><td>90.09</td></tr><tr><td>RXD178<sup>T</sup></td><td>83.37</td><td>87.08</td><td>86.90</td><td>84.37</td><td>84.85</td><td>100</td><td>83.32</td><td>83.37</td><td>86.60</td><td>84.40</td></tr><tr><td>Ter10<sup>T</sup></td><td>82.47</td><td>83.16</td><td>83.83</td><td>83.64</td><td>83.77</td><td>83.39</td><td>100</td><td>99.97</td><td>83.75</td><td>83.68</td></tr><tr><td>Ter282</td><td>82.48</td><td>83.20</td><td>83.80</td><td>83.67</td><td>83.79</td><td>83.39</td><td>99.97</td><td>100</td><td>83.75</td><td>83.73</td></tr><tr><td>Ter6<sup>T</sup></td><td>83.72</td><td>85.81</td><td>90.82</td><td>86.14</td><td>86.31</td><td>86.67</td><td>83.71</td><td>83.78</td><td>100</td><td>86.27</td></tr><tr><td>Ter91<sup>T</sup></td><td>85.02</td><td>83.95</td><td>85.74</td><td>98.14</td><td>90.18</td><td>84.37</td><td>83.72</td><td>83.74</td><td>86.34</td><td>100</td></tr></tbody></table><table><thead><tr><th colspan="11">ddH scores</th></tr><tr><th>Strain</th><th>Cal35</th><th>C3-17<sup>T</sup></th><th>H4R21<sup>T</sup></th><th>PMB3(1)</th><th>RLT1W51<sup>T</sup></th><th>RXD178<sup>T</sup></th><th>Ter10<sup>T</sup></th><th>Ter282</th><th>Ter6<sup>T</sup></th><th>Ter91<sup>T</sup></th></tr></thead><tbody><tr><td>Cal35</td><td>100</td><td>30</td><td>35.5</td><td>43.6</td><td>47.6</td><td>32.5</td><td>29.9</td><td>30</td><td>34.7</td><td>43.1</td></tr><tr><td>C3-17<sup>T</sup></td><td>30</td><td>100</td><td>37.4</td><td>32.4</td><td>34.7</td><td>40.9</td><td>32.3</td><td>32.4</td><td>36.9</td><td>32.2</td></tr><tr><td><strong>H4R21<sup>T</sup></strong></td><td><strong>35.5</strong></td><td><strong>37.4</strong></td><td><strong>100</strong></td><td><strong>43.8</strong></td><td><strong>43.9</strong></td><td><strong>45.8</strong></td><td><strong>36.9</strong></td><td><strong>37</strong></td><td><strong>60.2</strong></td><td><strong>43.8</strong></td></tr><tr><td>PMB3(1)</td><td>43.6</td><td>32.4</td><td>43.8</td><td>100</td><td>57.1</td><td>33.4</td><td>33.8</td><td>33.8</td><td>46.2</td><td>78.3</td></tr><tr><td>RLT1W51<sup>T</sup></td><td>47.6</td><td>34.7</td><td>43.9</td><td>57.1</td><td>100</td><td>36.5</td><td>35.2</td><td>35.3</td><td>41.5</td><td>55.7</td></tr><tr><td>RXD178<sup>T</sup></td><td>32.5</td><td>40.9</td><td>45.8</td><td>33.4</td><td>36.5</td><td>100</td><td>32.9</td><td>32.9</td><td>41.5</td><td>34.1</td></tr><tr><td>Ter10<sup>T</sup></td><td>29.9</td><td>32.3</td><td>36.9</td><td>33.8</td><td>35.2</td><td>32.9</td><td>100</td><td>100</td><td>34.1</td><td>33.5</td></tr><tr><td>Ter282</td><td>30</td><td>32.4</td><td>37</td><td>33.8</td><td>35.3</td><td>32.9</td><td>100</td><td>100</td><td>34.2</td><td>33.6</td></tr><tr><td>Ter6<sup>T</sup></td><td>34.7</td><td>36.9</td><td>60.2</td><td>46.2</td><td>41.5</td><td>41.5</td><td>34.1</td><td>34.2</td><td>100</td><td>47.9</td></tr><tr><td>Ter91<sup>T</sup></td><td>43.1</td><td>32.2</td><td>43.8</td><td>78.3</td><td>55.7</td><td>34.1</td><td>33.5</td><td>33.6</td><td>47.9</td><td>100</td></tr></tbody></table>
C. fungivorans, C. arenae and C. pratensis, but not in the genomes of the non-motile C. antrihumi, C. silvisoli and C. humicola. Colonies of strain H4R21T are transparent, circular and off-white with a circular margin around the main colony on 1/10 TSA incubated for 3 days at 25°C. Growth in LBm liquid medium occurred at 15-30°C (optimum at 20°C), pH 5-8 (optimum at pH 5) and with 0.5-2% NaCl. The chitin degradation assay revealed that strain H4R21T is able to hydrolyse chitin to the same extent as C. fungivorans Ter6T and C. pratensis PMB3(1), but better than C. arenae Ter10T and C. pratensis Ter91T, while no hydrolysis was observed for the reference strains of C. antrihumi, C. silvisoli and C. humicola. The siderophore production assay revealed that strain H4R21T is able to chelate iron as well as other tested strains (C. fungivorans, C. arenae, C. pratensis, C. silvisoli and C. humicola), except C. antrihumi C3-17T. The production of AHL was demonstrated for strain H4R21T as well as for strains of C. fungivorans (Ter6T, Ter331) and C. pratensis [Ter91T and PMB3(1)], for C. arenae (Ter282), Collimonas sp. strain Ca35, C. silvisoli (RXD178T) and C. humicola (RLT1W51T), but not for C. antrihumi [C3-17T] and C. arenae (Ter10T). Homologues of AHL synthase (i.e., luxI-like) and of the cognate transcriptional regulator LuxR were detected in the genomes of all AHL-producing strains, including H4R21T. Noticeably, strain H4R21T is able to consume D-mannitol, trehalose and N-acetyl-D-glucosamine, which are all known as fungal metabolites. D-Mannitol was not assimilated by C. antrihumi (C3-17T) and C. silvisoli (RXD178T) and trehalose not by C. humicola (RLT1W51T), C. arenae (Ter10T, Ter282) and Collimonas sp. strain Ca35. All other physiological and metabolic properties of H4R21T and Collimonas strains are shown in Table 2.

## CHEMOTAXONOMY

Cellular fatty acids of strain H4R21T were analysed by the DSMZ (Braunschweig, Germany) using biomass grown for 48 h on LBm at 25°C. Fatty acids were analysed as methyl esters by gas chromatography according to the instructions of the Microbial Identification System (MIDI Inc., USA) as described by Tindall [33, 34] and Kuykendall et al. [35].

The major respiratory quinone identified for strain H4R21T is ubiquinone 8 (Q-8), which is also found in the other Collimonas species [11, 12]. The polar lipid analyses revealed that strain H4R21T is characterized by phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, aminophospholipid, glycolipid, aminolipid, phospholipid and lipid. Diphosphatidylglycerol was also present in C. humicola and C. antrihumi, and variable among C. fungivorans strains and in C. silvisoli [11, 12] (Fig. S1 in the online Supplementary Material).

The major fatty acids (>5% of the total) of strain H4R21T were C$_{16:0}$ (35.3%), C$_{17:0}$ cyclo (6.6%), summed feature 3 (C$_{16:1}$ ω6c and/or C$_{16:1}$ ω7c; 35%) and summed feature 8 (C$_{18:1}$ ω7c and/or C$_{18:1}$ ω6c; 11.8%) (Table 3). C$_{16:0}$ was detected within the expected range of concentrations (33-40%) observed for the reference Collimonas strains. Noticeably, strain H4R21T had a lower relative

<caption>**Table 3.** Cellular fatty acid profiles of strain H4R21T and closely related type strains of the genus *Collimonas*</caption>

Strains: 1, C. rhizosphaerae H4R21T; 2, C. fungivorans Ter6T; 3, C. arenae Ter10T; 4, C. pratensis Ter91T; 5, C. antrihumi C3-17T; 6, C. silvisoli RXD178T; 7, C. humicola RLT1W51T. Data are expressed as percentages of the total fatty acids. The major components (>10% of the total) are highlighted in bold. ND, not detected. The data presented for the reference bacterial strains come from the literature.

<table><thead><tr><th>Fatty acid</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th></tr></thead><tbody><tr><td>C<sub>10:0</sub> 3-OH</td><td>0.6</td><td>0.9</td><td>1.0</td><td>0.9</td><td>1.1</td><td>1.1</td><td>0.8</td></tr><tr><td>C<sub>12:0</sub></td><td>4.9</td><td>3.4</td><td>4.1</td><td>3.4</td><td>3.6</td><td>3.4</td><td>3.8</td></tr><tr><td>C<sub>12:0</sub> 2-OH</td><td>1.4</td><td>2.5</td><td>1.9</td><td>2.4</td><td>2.5</td><td>2.2</td><td>2.0</td></tr><tr><td>C<sub>12:0</sub> 3-OH</td><td>2.1</td><td>3.1</td><td>3.4</td><td>3.7</td><td>3.8</td><td>3.3</td><td>3.3</td></tr><tr><td>C<sub>14:0</sub></td><td>0.8</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>C<sub>16:0</sub></td><td>35.3</td><td>40.2</td><td>40.8</td><td>40.2</td><td>33.4</td><td>35.6</td><td>38.5</td></tr><tr><td>C<sub>16:1</sub> 2-OH</td><td>ND</td><td>1.4</td><td>1.9</td><td>2.4</td><td>4.4</td><td>2.5</td><td>2.5</td></tr><tr><td>C<sub>17:0</sub> cyclo</td><td>6.6</td><td>24.1</td><td>17.0</td><td>21.5</td><td>13.1</td><td>19.4</td><td>21.7</td></tr><tr><td>iso-C<sub>17:0</sub> 3-OH</td><td>ND</td><td>2.8</td><td>ND</td><td>2.3</td><td>1.9</td><td>2.4</td><td>2.5</td></tr><tr><td>C<sub>18:0</sub></td><td>1.2</td><td>1.3</td><td>1.7</td><td>0.7</td><td>1.5</td><td>1.6</td><td>ND</td></tr><tr><td>Summed feature 3*</td><td>35</td><td>15.4</td><td>9.6</td><td>18.8</td><td>22.6</td><td>12.7</td><td>14.2</td></tr><tr><td>Summed feature 8*</td><td>11.8</td><td>4.5</td><td>6.8</td><td>7.2</td><td>9.6</td><td>13.2</td><td>7.6</td></tr></tbody></table>

<footnote>*Summed features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed feature 3, C$_{16:1}$ ω7c and/or C$_{16:1}$ ω7t; summed feature 8, C$_{18:1}$ ω7c and/or C$_{18:1}$ ω7t.</footnote>
abundance of C₁₇:₀ cyclo (6.6% in strain H4R21T vs 13–24% in other *Collimonas*) and a higher relative abundance of summed feature 3 (35% in strain H4R21T vs 9.6–22.6% in other *Collimonas*) [11, 12], making this strain distinguishable from the other *Collimonas* species.

## DESCRIPTION OF COLLIMONAS RHIZOSPHAERAE SP. NOV.

*Collimonas rhizosphaerae* (rhi.zo.sphae'rae. Gr. fem. n. *rhiza*, a root; Gr. n. *sphaira*, a ball, a sphere; N.L. fem. n. *rhizosphaera*, the rhizosphere; N.L. gen. fem. n. *rhizosphaerae*, of the rhizosphere, referring to the source of isolation of the type strain).

Colonies on 1/10 TSA medium are transparent, circular and slightly white with a differentiation of a circular margin around the main colony. Growth requires 3 days on LBm, 1/10 TSA and ABm media. No pigment is observed on these media. Tolerates up to 2% NaCl and grows at temperatures between 15 and 30°C, with an optimum temperature of 20°C. Growth occurs at pH 5–8 and the optimum is pH 6. Positive for the hydrolysis of colloidal chitin and siderophore production. The following carbon sources are used: 2-aminoethanol, α-D-glucose, α-ketobutyric acid, α-ketoglutaric acid, β-hydroxybutyric acid, bromosuccinic acid, *cis*-aconitic acid, citric acid, D-gluconic acid, D-glucuronic acid, D-glucosaminic acid, D-glucose-6-phosphate, D-psicose, D-sorbitol, trehalose, D-alanine, D-arabitol, D-fructose, D-galactonic acid lactone, D-galactose, D-mannitol, D-mannose, D-saccharic acid, DL-α-glycerol phosphate, DL-carnitine, DL-lactic acid, dextrine, formic acid, γ-aminobutyric acid, glucuronamide, glycerol, glycogen, glycyl-L-aspartic acid, glycyl-L-glutamic acid, inosine, L-alanine, L-alainamide, L-allanyl-Glycine, L-arabinose, L-asparagine, L-aspartic acid, L-fucose, L-glutamic acid, L-histidine, L-leucine, L-ornithine, L-proline, L-pyroglutamic, L-serine, L-threonine, *m*-inositol, malonic acid, *N*-acetyl-D-glucosamine, phenylethylamine, propionic acid, pyruvic acid methyl ester, quinic acid, succinamic acid, succinic acid, Tween 40, Tween 80, uridine and xylitol. In contrast, strain H4R21T is unable to assimilate: 2,3 butanediol, α-cyclodextrin, α-D-glycerol-1-phosphate, lactose, α-hydroxybutyric acid, α-ketovaleric acid, acetic acid, adonitol, methyl β-D-glucoside, D-galacturonic acid, raffinose, cellobiose, melibiose, D-serine, γ-hydroxybutyric acid, gentibiose, hydroxy-L-proline, *i*-erythritol, itaconic acid, L-phenylalanine, L-rhamnose, lactulose, maltose, *N*-acetyl-D-galactosamine, *p*-hydroxyphenylacetic acid, putrescine, sebacic acid, succinic acid monomethyl ester, sucrose, thymidine, turanose and urocanic acid are not assimilated. The predominant quinone is ubiquinone-8 (Q8). The major polar lipids are diphosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and lipid. Major fatty acids (>10%) are C₁₂:₀, C₁₂:₀ 3-OH, C₁₆:₀ and C₁₇:₀ cyclo.

The type strain is H4R21T (=DSM 117599T = CFBP 9203T), isolated from beech rhizosphere collected from the forest experimental site of Montiers (Meuse, France). The whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number JBANDC000000000. Raw data is available from the SRA database with accession number PRJNA1081282.

## Funding information

This work was supported by research funding from Novozyme to JHJL and by the French National programme EC2CO (Ecosphère Continentale et Côtière) and the Labex ARBRE to S.U. The UMR1136 and UR1138 programmes are supported by the ANR through the Laboratory of Excellence ARBRE (ANR-11-LABX-0002-01).

## Acknowledgements

We thank M-P. TURPAULT from INRAE, the National Office of Forests (ONF), and ANDRA for the access to the Montiers experimental site ('toposequence N°2'). We also thank the colleagues from the DSMZ and CIRM-CFBP for their support in strain deposit.

## Author contributions

S.U. extracted genomic DNA and performed the laboratory experiments. S.B. and M.B. contributed to the biochemical characterization. F.A. contributed to the genomic DNA conditioning. R.K., M.W.R. and J.P. generated the genome sequence and assembly. E.M. performed the phylogenomic and bioinformatic analyses. S.U. analysed the data and drafted the manuscript. S.U. and J.H.L. designed the experiments and revised the manuscript.

## Conflicts of interest

The authors declare that there are no conflicts of interest.

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