Hyphomonas sediminis sp. nov., isolated from marine sediment

doi:10.21203/rs.3.rs-1608969/v1

A Gram-staining-negative, aerobic and pear-shaped bacterial strain, designated WL0036^T, was isolated from coastal sediment sample collected in Nantong city, Jiangsu province of China (120°51′13″E, 32°6′26″N) in October 2020. Strain WL0036^T was found to grow at 20–37°C (optimum, 28°C) with 0–9.0% NaCl (optimum, 2.5-4.0%) and displayed alkaliphilic growth with the pH range of pH 6.0–10.0 (optimum, pH 7.0–8.0). The polar lipids profile of strain WL0036^T included phosphatidylcholine, phosphatidylethanolamine, glycolipid and an unidentified lipid. The major isoprenoid quinone was determined to be Q-11 and the major fatty acids were C_16:0, 11-methyl-C_18:1ω7c, and summed features 8 (C_18:1ω6c and/or C_18:1ω7c). The G + C content of genomic DNA was 61.8%. Phylogenetic trees constructed based on 16S rRNA gene sequence and bac120 gene set indicted that strain WL0036^T clustered with strains Hyphomonas neptunium ATCC 15444^T and H. polymorpha PS728^T. The average nucleotide identities between strain WL0036^T and strains H. neptunium ATCC 15444^T and H. polymorpha PS728^T were 80.7% and 81.2%, respectively. Strain WL0036^T showed 22.8% and 23.2% of digital DNA-DNA hybridization identities with H. neptunium ATCC 15444^T and H. polymorpha PS728^T, respectively. As inferred from the phenotypic and genotypic characteristics and the phylogenetic trees, strain WL0036^T ought to be recognized as a novel species in genus Hyphomonas, for which the name Hyphomonas sediminis sp. nov. is proposed. The type strain is WL0036^T (= MCCC 1K05843^T = JCM 34658^T = GDMCC 1.2413^T).

Hyphomonas sediminis sp. nov.

marine sediment

bac120 tree

The genus Hyphomonas was originally proposed by Pongratz (Pongratz 1957). The cells of the genus Hyphomonas were Gram-staining-negative, aerobic, heterotrophic and sporeless. The daughter cells are oval to pear-shaped, smaller than the body of the mother cell, moving through polar lateral flagella. Hyphae rarely branches during normal growth (Pongratz 1957). At the time of writing, the genus Hyphomonas includes 12 species, of which 11 have been validly published according to the List of Prokaryotic names with Standing in Nomenclature (LPSN) server (https://lpsn.dsmz.de/genus/hyphomonas, accessed on March 30, 2022) (Parte 2018): H. polymorpha (Moore et al. 1984); H. neptunium (Moore et al. 1984); H. oceanitis (Weiner et al. 1985); H. jannaschiana (Weiner et al. 1985); H. adhaerensand (Weiner et al. 2000a); H. johnsonii (Weiner et al. 2000a); H. atlanticus (Li et al. 2014c); H. beringensis (Li et al. 2014a); H. pacifica (Li et al. 2016), H. rosenbergii (Weiner et al. 2000b) and H. chukchiensis (Li et al. 2014a). Nevertheless, H. rosenbergii was recommended to be place on the reject list because the 16S rRNA gene sequence of H. rosenbergii ATCC 43869^T shared a similarity of 92.8% with the primitive sequence (Lai et al. 2015; Li et al. 2014b). Members of the genus Hyphomonas are primarily spread in the marine environment, which include pelagic areas, offshore areas and deep-sea thermal springs.

To investigate the application potential of the bacteria from offshore areas of Jiangsu province, China, a novel bacterium, designated WL0036^T, was isolated from a marine sediment sample of Nantong city. In the present study, we conducted a polyphasic taxonomic study to clarify the taxonomic position of strain WL0036^T.

Isolation and culture condition

Strain WL0036^T was isolated from a marine sediment sample collected in Nantong city, Jiangsu province of China (120^o51′13″E, 32^o6′26″N) in October 2020. Prior to isolation, the sample was stored in -4^oC refrigerator. For isolation, 2 g sample was diluted in sterile physiological saline (18 mL), and then incubated in a constant temperature shaker incubator at 28^oC for 1 h. The suspension was diluted to 10^− 3 and 100 µL were spread on plates of the marine agar medium 2216E (per litre: 5.0 g peptone, 1.0 g yeast extract, 15.0 g agar, 0.01 g ferric phosphate, 25 g sea salt, 0.5 g NaOH). After incubation at 28^oC for 5 days, colonies were picked up and streaked on tryptose soya agar (TSA, per litre: 15.0 g tryptone, 5.0 g soy papain digest, 5.0 g sodium chloride, 15.0 g agar) medium supplemented with 2.5% sea salt to obtain pure cultures. Subsequently, the bacterium was maintained on TSA (Difco) medium supplemented with 2.5% sea salt and also stored at -80^oC in 20.0% (v/v) glycerol.

Genome sequencing and annotation

For 16S rRNA gene amplification by PCR, genomic DNA was extracted by using the Ezup column bacterial genomic DNA purification kit (Sangon Biotech, China). The crude extracts were used as DNA template for PCR as described previously (Weisburg et al. 1991) with 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-AAGGAGGTGATCCAGCC-3′) primers. The almost full-length 16S rRNA gene sequence was compiled using SeqMan software (DNASTAR) and analyzed through the EzbioCloud (https://www.ezbiocloud.net/) server (Yoon et al. 2017) to retrieve sequences of the most closely related types strains. Whole-genome shotgun sequencing of strain WL0036^T was carried out by Magigene company, Guangzhou, China. The whole genome was assembled using SPAdes version 3.10.1 (Nurk et al. 2013) in UGENE software package (Okonechnikov et al. 2012). Following the proposed minimal standards for the use of genome data for the taxonomy of prokaryote (Goris et al. 2007; Richter 2009), the average nucleotide identify (ANI) and the digital DNA-DNA hybridization (dDDH) were used to compare strain WL0036^T and related type species genome sequences. The ANI values was calculated by ANI calculator using the OrthoANIu algorithm on the EzBioCloud website (https://www.ezbiocloud.net/) (Yoon et al. 2017) and the dDDH values was calculated using the Genome-to-Genome Distance Calculator (http://ggdc.dsmz.de/ggdc.php/) (Meier-Kolthoff et al. 2013). Genome annotation was conducted by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al. 2016), eggNOG online server (https://eggnog-mapper. embl.de/) (Huerta-Cepas et al. 2017) and KEGG online server (https://www.kegg.jp/blastkoala/) (Kanehisa et al. 2016), The results of genetic analysis for drug resistance was obtained through the Comprehensive Antibiotic Resistance Database (CARD) website (https://card.mcmaster.ca/) (Alcock et al. 2020). The G + C content of genomic DNA was calculated based on the EzBioCloud server from the draft genome sequence.

Phylogenetic analysis

Phylogenetic trees based on 16S rRNA gene sequence were reconstructed by the neighbor-joining (NJ) (Saitou and Nei 1987), minimum-evolution (ME) (Pardi et al. 2010), and maximum-parsimony (MP) (Fitch 1971) methods using MEGA X software (Kumar et al. 2018) after multiple sequence alignments. The evolutionary distance matrix of the phylogenetic tree was commonly calculated using Kimura's two-parameter model (Kimura 1980). Confidence values for tree branches were determined by bootstrap analysis based on 1000 resamples (Felsenstein 1985). Genome-based phylogenies of supermatrix and supertree approaches from protein sequences of the bac120 gene set were constructed by using EasyCGTree version 3.0 (https://github.com/zdf1987/EasyCGTree) as described previously (Zhang et al. 2020). The genome sequences of the strains of interest were downloaded from the NCBI genome database (https://www.ncbi.nlm.nih.gov/genome/) and detailed information is listed in Table S1.

Morphology, physiology, and biochemical analysis

A Gram Stain kit (Solarbio, China) was used for testing the Gram reaction. The cell morphology of the bacterium was observed by transmission electron microscopy (JEM, 2100) using cells grown in TSA (Difco) medium supplemented with 2.5% sea salt for 48 h at 28°C. Motility was determined by observing growth of cells in test tubes containing TSB medium with 0.3% agar after 3 days of incubation at 28^oC. Anaerobic growth was tested in an anaerobic chamber for 10 days on TSA medium supplemented with 2.5% sea salt. To determine the development conditions of the strain WL0036^T, growth at various temperatures (4, 10, 20, 28, 37, 45, and 50°C) was tested in TSA medium supplemented with 2.5% sea salt. Adjusting pH values (pH 4.0–12.0, with 1.0 increments), using NaOH (1.0%, w/v) and HCl (1.0%, v/v) buffer systems to adjust pH in the tryptic soy broth (TSB, per litre: 17.0 g tryptone, 5.0 g sodium chloride, 3.0 g soy papain digest, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose (monohydrate/anhydrous), pH 7.3 ± 0.2, 25°C) medium. To investigate the tolerance to NaCl, growth at various NaCl concentrations (0, 1.5, 2.5, 4.0, 7.0, 9.0, 11.0 and 13.0%) was investigated in TSA medium.

Catalase activity was detected by observing whether bubbles were produced using 3.0% hydrogen peroxide. Oxidase activity was determined with the use of 1.0% tetramethyl-β-phenylenediamine. Additional biochemical tests were performed using the BIOLOG GEN III microtest system (Biolog, USA), API 20NE and API ZYM systems (bioMérieux, France) in addition to adjusting the salinity to 2.5% with sea salt when culturing strain WL0036^T according to the manufacturer's instructions.

Chemotaxonomic characterization

The strains were incubated in TSB medium supplemented with 2.5% sea salt at 28°C for three days, and then the biomass was collected for fatty acid characterization. Cellular fatty acids was methylated and analyzed using the Sherlock Microbial Identification System (MIDI) according to a previously described method and the manufacturer’s instructions (Sasser 1990). The polar lipid profile and isoprenoid quinones were determined using cells grown on TSB medium containing 2.5% sea salt at 28°C for 3 days. Two-dimensional thin-layer chromatography was performed following previously demonstrated methods to detect polar lipids in cells (Collins and Jones 1980; Minnikin et al. 1984). Phosphomolybdic acid was used for determination of all polar lipids, ninhydrin for phospholipids containing amino or glucosamine, and anisaldehyde and α-naphthol were used for glycolipids. Isoprenoid quinones were extracted (Collins et al. 1977) and analysed using reversed phase HPLC as described previously (Tamaoka 1986).

Phylogenetic analysis

The results from the EzBioCloud server suggested that the 16S rRNA gene sequence of strain WL0036^T exhibited the highest similarities to those of H. polymorpha PS728^T (98.3%) and H. neptunium ATCC 15444^T (97.8%). By using the neighbor-joining method, the phylogenetic tree (Fig. 1) denoted that strain WL0036^T clustered with H. polymorpha PS728^T, H. rosenbergii VP-6^T and H. neptunium ATCC 15444^T, and the clade of these four taxa was closely related to the rest members of the genus Hyphomonas. Meanwhile, the phylogenetic position of strain WL0036^T was well supported (bootstrap value > 70.0%) in the neighbor-joining tree, and it agreed with the trees of maximum-parsimony method (Fig. S1) and the minimum-evolution method (Fig. S2) with high confidence (bootstrap values > 70).

In order to further explore the relationships among strain WL0036^T and related species within the genus Hyphomonas, the bac120 tree constructed following the method of the supermatrix (Fig. 2) demonstrated that strain WL0036^T formed a clade and clustered with the reference strains H. polymorpha PS728^T and H. neptunium ATCC 15444^T. Meanwhile, the phylogenetic tree based on 119 universal protein coding genes of the bac120 set among the strains of interest (Fig. S3) showed that strain WL0036^T also formed a clade with H. polymorpha PS728^T and H. neptunium ATCC 15444^T, which was supported by 113 of the 119 genes (95.0%) used in supertree method. This result was corresponding to that of the supermatrix method and those based on 16S rRNA gene sequence. Consequently, these findings support that strain WL0036^T was a member of genus Hyphomonas.

Genomic features

The draft genome of strain WL0036^T was comprised of 4 contigs with a size of 3,498,010 bp. The G + C content of genomic DNA was 61.8%. Automated annotation by NCBI PGAP recognized 3302 protein-coding sequences, 6 rRNA genes, 41 tRNA genes and 3 RNA genes of other type.

The annotation of KEGG indicated that strain WL0036^T had 12 genes involved in sulfur metabolism (ko00920): K1X12_RS02220 (cysI), K1X12_RS02210 (cysH), K1X12_RS12225 (cysE), K1X12_RS05225 (metA), K1X12_RS07770 (cysN), K1X12_RS07775 (cysD), K1X12_RS10865 (sseA), K1X12_RS04790 (cysQ), K1X12_RS03670 (cysK), K1X12_RS00130 (tauD), K1X12_RS16560 (metZ), K1X12_RS14335 (sqr), of which K1X12_RS02220 (cysI), K1X12_RS02210 (cysH) and K1X12_RS07770 (cysN) may confer assimilatory sulfate reduction; 27 genes were involved to flagellar assemblies (ko02040), of which 10 were involved in coding flagellar hook-basal body and flagellar basal body complex protein, and cells of strain WL0036^T with a single polar flagella (Fig. S4); Only 1 gene involved to carotenoid biosynthesis (ko00906) was K1X12_RS00430 (crtB). The annotation of CARD indicated that all three strains possessed the adeF, which could confer the active cell membrane efflux of fluoroquinolone antibiotics. And in the BIOLOG GEN III tests after 48 h of incubation at 28°C, all three strains were resistant to nalidixic acid (Table S2).

The ANI values between strain WL0036^T and strains H. neptunium ATCC 15444^T and H. polymorpha PS728^T were 80.7% and 81.2%, respectively (Table 1), all of which the ANI values were below threshold for species boundaries (95.0%-96.0%) (Richter 2009). Strain WL0036^T showed dDDH values of 22.8% and 23.2% with H. neptunium ATCC 15444^T and H. polymorpha PS728^T, respectively (Table 1). The dDDH values were far below the species delineation threshold 70% (Goris et al. 2007). These data illustrated that strain WL0036^T represents a novel species.

Table 1

**The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analysis among WL0036**^T **and closely related species.**
Strain	1	2	3
1	-	80.72	81.24
2	22.80	-	82.93
3	23.40	25.00	-
Taxa: 1, WL0036^T; 2, Hyphomonas neptunium MCCC 1A09418^T; 3, Hyphomonas polymorpha DSM 2665^T.
ANI values are displayed below the diagonal and dDDH values are displayed above the diagonal.
The values presented in the table are in the form of percentages.

Phenotypic characteristics

The cells of strain WL0036^T were Gram-staining-negative, aerobic, motile and pear-shaped with a single polar flagellum. Slight yellow, round and smooth colonies were observed on TSA medium with 2.5% sea salt for 3 days at 28°C. More results on phenotypic characteristics were listed in Table 2 and Table S2.

Table 2

Differential characteristics between strain WL0036^T and closely related species.
Characteristic	1	2	3
Colony colour	Slight yellow	White	White
Motility	+	+	+
Temperature range for growth (^oC) (optimum)	20–37 (28)	20–37 (28)	10–37 (20–28)
pH range for growth (optimum)	6.0–10.0 (7.0–9.0)	6.0–10.0 (7.0–9.0)	6.0–8.0 (7.0)
NaCl tolerance (%) (optimum)	0–9.0 (1.5-4.0)	0–9 (1.5-4.0)	0–9.0 (1.5-4.0)
G + C content (mol%)	61.8	61.9	62.3
Major respiratory Quinone	Q-11	Q-11	Q-11
API ZYM test
N-Acetyl-β-glucosaminidase	-	-	+
α-Chymotrypsin	+	-	-
α-Fucosidase	-	+	-
α-Glucosidase	-	+	-
Trypsin	-	+	+
Valine arylamidase	-	+	+
API 20NE test
4-Nitrophenyl β-D-galactopyranoside (PNPG)	-	-	+
Taxa: 1, WL0036^T; 2, Hyphomonas neptunium ATCC 15444^T; 3, Hyphomonas polymorpha PS728^T. +, positive; -, negative. All data from this study.
API 20NE test, enzyme assays for strain WL0036^T, Hyphomonas neptunium ATCC 15444^T and Hyphomonas polymorpha PS728^T indicated that all strains were negative for arginine dihydrolase, D-glucose fermentation, hydrolysis of gelatin, hydrolysis of urea, nitrate reduction, positive for hydrolysis of esculin and indole production. All strains were positive for catalase and oxidase.
Assimilation tests of strain WL0036^T, Hyphomonas neptunium ATCC 15444^T and Hyphomonas polymorpha PS728^T were all negative for utilization of N-acetylglucosamine, adipic acid, L-arabinose, capric acid, D-glucose, malic acid, D-maltose, D-mannitol, D-mannose, phenylacetic acid, potassium gluconate, trisodium citrate.
API ZYM test, strain WL0036^T, Hyphomonas neptunium ATCC 15444^T and Hyphomonas polymorpha PS728^T were all negative for acid phosphatase, alkaline phosphatase, cystine arylamidase, esterase (C4), esterase lipase (C8), β-glucosidase, leucine arylamidase and naphthol-AS-BI-phosphohydrolase, positive for α-galactosidase, β-galactosidase, β-glucuronidase, lipase (C14) and α-mannosidase.

Chemotaxonomic characteristics

Strain WL0036^T exhibited a polar lipid profile consisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE), glycolipid (GL) and an unidentified lipid (L) (Fig. S5). The predominant cellular fatty acids (> 10%) of strain WL0036^T were C_16:0 (27.6%), 11-methyl-C_18:1ω7c (12.1%), and summed features 8 (C_18:1ω6c and/or C_18:1ω7c) (35.0%) (Table. S3). The compositions of cellular fatty acid contents (> 1%) of WL0036^T and the neighboring type strains were presented in Table S2. The predominant isoprenoid quinone was ubiquinone-11 (Q-11), which was identical to that of the two neighboring type strains.

Taxonomic conclusions

The phylogenetic analysis from 16S rRNA gene sequence and bac120 gene set suggested that strain WL0036^T should be a member of the genus Hyphomonas (Fig. 1, Fig. 2). Characterization by physiological and biochemical experiments showed that strain WL0036^T shared many common traits with the two neighboring type strains. However, strain WL0036^T was significantly distinguished from the two neighboring type strains in terms of its capability to utilize gelatin and L-arginine as carbon sources and its positive activity for α-chymotrypsin (Table. 2). Beside of the evidence above, strainWL0036^T also can be distinguished from H. neptunium ATCC 15444^T and H. polymorpha PS728^T by other phenotypic differences (Table. S2). For example, strain WL0036^T is negative for utilization of N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, D-cellobiose, gentiobiose, D-glucose-6-PO₄, D-malic acid, L-malic acid, D-melibiose, β-methyl-D-glucoside and L-rhamnose as carbon sources, positive for utilization of acetic acid, acetoacetic acid and α-keto-glutaric acid as carbon sources, negative for resistance of aztreonam, α-glucosidase, lincomycin, β-rockulosidase, D-serine, tetrazolium blue, troleandomycin, trypsin, valine aromatase and vancomycin, which can clearly distinguish the strain WL0036^T from the strain H. neptunium ATCC 15444^T; strain WL0036^T is positive for utilization of L-arginine, L-aspartic acid, D-fucose, L-fucose, D-fructose-6-PO₄, gelatin, glycyl- L-histidine and L-prolin as carbon sources, negative for utilization of L-butyric acid, L-lactic acid, D-malic acid, L-malic acid and methyl Pyruvate, negative for resistance of fusidic acid, troleandomycin, tetrazolium blue, lincomycin and vancomycin, which can clearly distinguish the strain WL0036^T from the strain H. polymorpha PS728^T.

Based on the results of phenotypic, phylogenetic, the ANI values and the dDDH values, strain WL0036^T was suggested to be clearly distinguished from H. polymorpha PS728^T and H. neptunium ATCC 15444^T. Consequently, strain WL0036^T was proposed as a novel species in the genus Hyphomonas, for which the name Hyphomonas sediminis sp. nov. is proposed.

Description of Hyphomonas sediminis sp. nov.

Hyphomonas sediminis [se.di’mi.nis. L. gen. neut. n. sediminis, of sediment].

Cells are Gram-staining-negative, aerobic, motile and pear-shaped. Cells grow well on TSA medium with 2.5% sea salt, marine agar medium 2216E. Growth occurs at 20°C-37°C (optimum 28 ^oC), pH 6.0–10.0 (optimum 7.0–8.0) and with 0%-9.0% NaCl (optimum 2.5%-4.0%). Cells are positive for both oxidase and catalase. The strain showed negative reactions (API 20NE) in the assimilation of L-arabinose, N-acetylglucosamine, capric acid, D-glucose, malic acid, D-maltose, D-mannose, D-mannitol, potassium gluconate, phenylacetic acid and trisodium citrate, showed positive reactions (API 20NE) in the enzyme assays of hydrolysis of esculin and indole production, negative for arginine dihydrolase, D-glucose fermentation, hydrolysis of gelatin, hydrolysis of urea, nitrate reduction and 4-nitrophenyl β-D-galactopyranoside (PNPG). In the API ZYM tests, positive for chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, lipase (C14) and α-mannosidase, but negative for N-acetyl glucosaminidase, acid phosphatase, alkaline phosphatase, cystine aramase, esterase (C4), esterase lipase (C8), α-glucosidase leucine arylamidase, β-rockulosidase, naphtol-AS-BI-phosphohydrolase, trypsin, β-uronic acid glycase and valine aramase. In the BIOLOG GEN III tests, positive for utilization of L-arginine, L-aspartic acid, L-butyric acid D-fructose-6-PO₄, D-fucose, L-fucose, gelatin, L-glutamic acid, glycyl-L-prolin, L-histidine, β-hydroxy-D, L butyric acid, α-keto-glutaric acid, L-serine and tween 40, and is resistant to lithium chloride, nalidixic acid, potassium tellurite, rifamycin SV and sodium butyrate; the rest tests are all negative. The polar lipid profile consists of phosphatidylcholine, phosphatidylethanolamine, glycolipid and an unidentified lipid. Q-11 is the sole respiratory quinone. The major cellular fatty acids (> 10%) are C_16:0, 11-methyl-C_18:1ω7c, and summed features 8 (C_18:1ω6c and/or C_18:1ω7c).

The type strain, WL0036^T (= MCCC 1K05843^T = JCM 34658^T = GDMCC 1.2413^T), the DNA G + C content of genomic DNA is 61.8%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the draft genome sequence of strain WL0036^T are OL605964 and JAIEZP000000000, respectively.

Author’s contributions

DFZ and WJL designed research and project outline. LW and DFZ performed isolation, deposition and polyphasic taxonomy. DFZ, LW and WH performed genome analysis. LW, WJL AHZ and DFZ drafted the manuscript. AHZ, WH, ZYG, JKH and CL revised the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Data availability statement

All of the data supporting the conclusions of this article are included within the article and its additional files. The genome datasets and the 16S rRNA gene sequence of Hyphomonas sediminis WL0036^T generated during the current study are available in the GenBank/EMBL/DDBJ repository under accession number JAIEZP000000000 and OL605964. Other datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. Other genome sequence data detailed information was listed in Table S1.

Funding details

This research was supported by the National Natural Science Foundation of China (No. 31900001), the China Postdoctoral Science Foundation (2020M671312) and the Fundamental Research Funds for the Central Universities (B210202140).

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No competing interests reported.

SupplementaryMaterial.docx

Hyphomonas sediminis sp. nov., isolated from marine sediment

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Isolation and culture condition

Genome sequencing and annotation

Phylogenetic analysis

Morphology, physiology, and biochemical analysis

Chemotaxonomic characterization

Results And Discussion

Phylogenetic analysis

Genomic features

Phenotypic characteristics

Chemotaxonomic characteristics

Taxonomic conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1