Halorussus halobius sp. nov., Halorussus marinus sp. nov. and Halorussus pelagicus sp. nov., isolated from salted brown alga Laminaria

Four halophilic archaeal strains, designated HD8-83 T , LYG-36 T , DLLS-82 and RC-68 T , were isolated from the salted brown alga Laminaria of three different origins (Dalian, Lianyungang, Dalian and Rongcheng) in China. All strains had pleomorphic rod cells that were motile, lysed in distilled water, stained Gram-negative and formed red-pigmented colonies on agar plate, except DLLS-82 showing white colonies. Based on phylogenetic analyses of the 16S rRNA genes, strain HD8-83 T was closely related to Halorussus litoreus HD8-51 T (97.9 % similarity), strain LYG-36 T and DLLS-82 were closely related to Halorussus rarus TBN4 T (94.4 % and 94.7 % similarities, respectively), and strain RC-68 T exhibit close to Halorussus salinus YJ-37-H T (96.9 % similarity). Phylogenetic analyses based on rpoB′ gene and 728 concatenated single-copy orthologous clusters also show these strains formed three different branches and clustered tightly with the Halorussus members. The ANI, AAI and isDDH values between strain LYG-36 T and DLLS-82 were 98.9 %, 98 % and 92.4 %, showing they were different strains of same species. While those values between isolates and other Halorussus members with values below 84.7 %, 82.9 % and 28.9 %. Based on the phenotypic, chemotaxonomic and phylogenetic properties, the strains HD8-83 T , LYG-36 T , DLLS-82 and RC-68 T represent three novel species of the genus Halorussus for which the names Halorussus halobius sp. nov. (type strain HD8-83 T = CGMCC 1.15334 T = JCM 31110 T ), Halorussus marinus sp. nov. (type strain LYG-36 T = CGMCC 1.13606 T = JCM 32952 T , reference strain DLLS-82 = CGMCC 1.13604 = JCM 32951) and Halorussus pelagicus sp. nov. (type strain RC-68 T = CGMCC 1.13609 T = JCM 32953 T ) are proposed. galactosyl mannosyl glucosyl diether (TGD-1) and diglycosyl diether (DGD-2). The DNA G + C content of strain RC-68 T is 63.8 mol%. The major respiratory quinones were menaquinone MK-8 and MK-8(H 2 ).


Introduction
In China, the brown alga Laminaria is the most important arti cial mariculture seaweed and most of these were used for making food (www.seaweed.ie).For annual kelp production was huge, salting, one of the oldest methods for food preservation, was mainly selected for processing and preserving Laminaria, which can extend shelf life by preventing spoilage and growth of microorganisms (Taormina 2010).
Meanwhile high concentration of sodium chloride becomes a nice habitat for halophilic archaea and bacteria, once they were taken from raw kelp or processing salt, then grow and multiply.According to our previous results, salted Laminaria inhabited by a dense population of haloarchaea (Wei et al. 2021).Members of haloarchaea require high salt concentrations to grow, most species like more than 2.0 M NaCl (Yadav et al. 2015), heterotrophic aerobically or anaerobically.
In the previous focusing on the halophilic archaeal diversity in salted kelp produced in different coastal regions of China, four halophilic archaeal strains were isolated and found to be most closely related to the members of Halorussus according 16S rRNA gene analysis.The genus Halorussus (Hrs.) was established based on two strains of Hrs.rarus (Cui et al. 2010), and now it comprises six validly published species, which were isolated from Taibei, Yangjiang and Zhoushan marine solar saltern (Hrs.rarus, Hrs.salinus and Hrs.halophilus), Yuncheng salt lake (Hrs.amylolyticus and Hrs.ruber), salted Laminaria (Hrs.litoreus) in China (Cui et Yuan et al. 2015b).In this study, the isolated four halophilic archaeal strains were characterized by describing phenotypic and phylogenetic properties then assigned into three novel species, Halorussus halobius sp.nov., Halorussus marinus sp.nov.and Halorussus pelagicus sp.nov.

Strains isolation and cultivation
The novel halophilic archaeal strains were isolated from brine samples of salted Laminaria produced in different coastal regions of China.Strain HD8-83 T and DLLS-82 were isolated from salted Laminaria produced in Daliang, Liaoning Province (pH 6.9 and salinity 293 g l -1 ), strain LYG-36 T from that produced in Lianyungang, Jiangsu Province, China (pH 6.3 and salinity 208 g l -1 ) and strain RC-68 T from that produced in Rongcheng, Shandong Province, China (pH 7.5 and salinity 285 g l -1 ).The serially diluted the brine was inoculated onto the neutral haloarchaeal medium (NHM) which contained (l - Cui 2020) .The agar plates were incubated at 37 °C for one month under aerobic condition.The single colonies were transferred and were successively re-streaked on NHM agar plates at least three times to obtain pure colonies.The isolated strains were preserved at −20 °C as a suspension in NHM broth supplemented with glycerol (150 g l -1 ).The isolated strains together with reference strains were routinely grown aerobically at 37 °C in NHM medium.

Whole-genome sequencing and phylogenetic analysis
The fresh haloarchaeal cultures grown in NHM liquid medium at 37 °C were used for genomic DNA extraction and purification using a genomic DNA extraction kit (CW0552, Beijing ComWin Biotech Co., Ltd.).The draft genomes of strain HD8-83 T , LYG-36 T , DLLS-82 were sequenced using an Illumina HiSeq PE150 platform (Novogene Co., Ltd, Beijing).
The library construction included A-tailed, ligated to paired-end adaptors and PCR amplified with a 350 bp insert.The reads were de novo assembled using SOAP de novo software (Luo et al. 2012) .The complete genome of strain RC-68 T was sequenced

Genomic analyses
The genomes were annotated through RAST server (Overbeek et al. 2014) .
The OrthoVenn2 online tool (Xu et al. 2019) was used to compare the OCs among the novel strains and their closely related species.

Morphological, physiological and chemotaxonomic characterization
The cellular morphological features were observed by Nikon microscopy (Ci-L) equipped with phase-contrast optics (model: E400) using cells grown in NHM broth at 37 °C for Halophilic archaeal polar lipids were extracted using a chloroform-methanol system and analyzed by oneand two-dimensional TLC according to Cui et al (Cui et al. 2010) .The general detection reagent, sulfuric acid-ethanol (1:2, by vol.), was used to detect total polar lipids.Isoprenoid quinones were extracted, purified by thin-layer chromatography.The isoprenoid quinones were confirmed by HPLC-MS analysis (Wainø et al. 2000) .

Results And Discussion
Phylogenetic and phylogenomic analyses The 16S rRNA gene sequence obtained both by conventional cloning Sanger sequencing and extracted from genome, showing that strain RC-68 T had three 16S rRNA genes, two of them were completely consistent, anther had 5 bp miss match (total 1472 bp).Strain HD8-83 T , LYG-36 T , DLLS-82 had one kind of 16S rRNA gene (1472 bp, 1472 bp, and 1471 bp, respectively).The highest 16S rRNA gene sequence similarities between each novel strain and its related Halorussus species are as following, 97.9 % similarity between strain HD8-83 T and Hrs.litoreus HD8-51 T , 94.4 % and 94.7 % similarities between strains LYG-36 T , DLLS-82 and Hrs.rarus TBN4 T , 96.9 % similarity between strain RC-68 T and Hrs.
A phylogenetic tree based on the 16S rRNA gene sequences of isolated strains and their closest neighbours is shown in Fig. 1a.Four strains grouped together with members of genus Halorussus, strain HD8-83 T was located in a separated branch closely related to Hrs. litoreus HD8-51 T ; strain LYG-36 T and DLLS-82 clustered together in one branch with similarity of 99.5% and was close to Hrs. rarus TBN4 T and Hrs.ruber YC25 T ; strain RC-68 T exhibit close phylogenetic relatedness to Hrs. salinus YJ-37-H T .The phylogenetic position was also con rmed in other trees generated using the MP and NJ algorithms.The 16S rRNA sequence similarity data and phylogenetic tree of the isolated strains support the establishment of three novel species placement in the genus Halorussus.Phylogenetic tree reconstruction based on rpoB′ gene indicate the same taxonomic a liation of the new strains (Fig. 1b).Strain HD8-83 T , LYG-36 T , DLLS-82 and RC-68 T tightly clustered with the members of Halorussus but formed independent branchs, clearly separated from the other genus.Strain HD8-83 T was phylogenetically relatedness to Hrs. rarus TBN4 T (92% similarity), strain LYG-36 T clustered with DLLS-82 and formed a separated branch, strain RC-68 T eas close to the clade consisted by Hrs.ruber YC25 T and Hrs.salinus YJ-37-H T (92.8% and 93.8% similarity, respectively).The phylogenetic position was also con rmed in trees generated using the maximum-parsimony (MP) and neighbour-joining (NJ) algorithms.
The phylogenomic tree reconstruction based on concatenated aligned single-copy OCs (Fig. 1c) showed that strain LYG-36 T together with DLLS-82 formed a distinct clade, which is closest to Hrs. amylolyticus YC93 T , strain HD8-83 T and RC-68 T form two separate branch closest to Hrs. litoreus HD8-51 T and Hrs.salinus YJ-37-H T , respectively.This result support that the four strains belong to three novel species within the genus Halorussus.
Strain RC-68 T together with the closest strain YJ-37-H T required higher concentration of Mg 2+ (0.3 M) for optimum growth than the others wanted (0.005-0.1 M).The abilities of anaerobic growth, indole formation, H 2 S formation, gelatin hydrolysis, casein hydrolysis and oxidase were strain-dependent.More detailed results of phenotypic features are given in the species description.

Genome characteristics
Three genomes and one complete genome of new strains ranged from 3.5-3.9Mbp (more genomic features in Table S1), slightly lower than the most genome size of Halorussus type strains ( The genomes of strains HD8-83 T , LYG-36 T and DLLS-82 had a single rRNA operon (one 5S rRNA, one 16S rRNA and one 23S rRNA gene), tRNA genes 51, 72, 49 and sRNA genes 3, 5, 5, respectively.The genome of strain RC-68 T had three rRNA operon (three 5S rRNA, three 16S rRNA and three 23S rRNA gene, located on the chromosome), tRNA genes 50 and sRNA genes 9 (Table S1).The heatmap based on gene counts of subsystem feature annotated by RAST server revealed that amino acids and derivatives, protein metabolism were the most abundant RAST subsystems for Halorussus members (Fig. S3), which suggest diverse metabolic functions in Halorussus.The clustering relationship of the number of annotation genes indicates the similarity of functional genes and the similarity of strain functions.For example, strains LYG-36 T and DLLS-82 are the same species, and their physiological and biochemical characteristics are also the most similar with each other.Strains LYG-36 T and DLLS-82 have the largest number of amino acids and derivatives functional genes (223 and 224), and they can use a variety of amino acids (glycine, L-alanine, L-arginine, L-aspartate, L-glutamate, L-lysine and L-ornithine), while strain RC-68 T has only 184 amino acids and derivatives functional genes, it also has the ability to use almost the same amino acid except glycine as the sole carbon source or nitrogen source for growth, which may be due to insu cient gene function annotation caused by imperfect metabolic pathway analysis of haloarchaea.
Comparison of the OCs among the novel type strains and Halorussus members revealed that all the species had 5322 clusters, including 3688 accessory OCs (shared with more than two but not all strains), 1716 core OCs (shared with all strains) and 104 unique (strain-speci c) clusters.Strain HD8-83 T contained 9 clusters, 2 of them annotated by GO datebase showing the function of mitotic DNA replication checkpoint and ATPase activity, strain LYG-36 T contained 20 unique clusters (4 had GO annotation: nicotine catabolic process, nucleotide binding, RNA-directed DNA polymerase activity, mitotic DNA replication checkpoint), strain RC-68 T contained 4 unique clusters (2 had GO annotation: structural molecule activity and DNA restriction-modi cation system), other strains had various number of unique clusters (Fig. S4).These results indicated that strains HD8-83 T , LYG-36 T and RC-68 T were different from each other and their closely related species.

Conclusions
The low level of 16S rRNA gene sequence similarity, ANI, AAI and DDH values, the independent phylogenetic position, and the differences in phenotypic properties and chemotaxonomic characteristics between strain HD8-83 T , LYG-36 T , DLLS-82, RC-68 T and its closest phylogenetic described species of Halorussus indicated that these strains diverged from current members.Therefore, we suggest that the strains HD8-83 T , LYG-36 T , DLLS-82, and RC-68 T represents three novel species of the genus Halorussus Halorussus halobius (ha.lo'bi.us.Gr. n. hals, halos salt; Gr. n. bius life; M.L. masc.adj.halobius living on salt).
Cells lyse in distilled water and the minimal NaCl concentration to prevent cell lysis is 0.85 M. The catalase activity and oxidase activity are positive.Anaerobic growth is observed in the presence of nitrate, with nitrate reduction to nitrite but no gas formation from nitrate.Anaerobic growth with DMSO and Larginine is not detected.Indole formation and H 2 S formation are negative.The type strain can not hydrolyze gelatin, hydrolyze casein, starch or Tween 80.The following substrates are utilized as single carbon and energy sources for growth: D-glucose, D-galactose, maltose, sucrose, glycerol, D-mannitol, Dsorbitol, pyruvate, DL-lactate, succinate, L-malate and citrate.The following substrates are utilized as single carbon, nitrogen or energy sources for growth: glycine, L-arginine, L-aspartate, L-glutamate and Lornithine.D-mannose, D-fructose, L-sorbose, D-ribose, D-xylose, lactose, starch, acetate or fumarate cannot be utilized as single carbon and energy sources for growth.L-alanine and L-lysine cannot be utilized as single carbon, nitrogen or energy sources for growth.
The DNA G + C content of strain HD8-83 T is 67.3 mol%.The type strain is HD8-83 T (= CGMCC 1.15334 T = JCM 31110 T ) and was isolated from salted brown alga Laminaria produced at Dalian, Liaoning Province, China.
Description of Halorussus marinus sp.nov.
Description of Halorussus pelagicus sp.nov.
The catalase activity is positive and the oxidase activity is negative.Anaerobic growth is not observed in the presence of nitrate, DMSO and L-arginine, and not detected nitrate reduction to nitrite or gas formation from nitrate.Indole formation and H 2 S formation are positive.The type strain can hydrolyze gelatin and casein, but not hydrolyze starch or Tween 80.The following substrates are utilized as single carbon and energy sources for growth: D-glucose, D-mannose, D-galactose, sucrose, lactose, glycerol, Dmannitol, D-sorbitol, acetate, pyruvate, DL-lactate, succinate, L-malate, fumarate and citrate.The following substrates are utilized as single carbon, nitrogen or energy sources for growth: L-alanine, Larginine, L-aspartate, L-glutamate, L-lysine and L-ornithine.D-fructose, L-sorbose, D-ribose, Dxylose,maltose or starch cannot be utilized as single carbon and energy sources for growth.Glycine cannot be utilized as single carbon, nitrogen or energy sources for growth.
The type strain is RC-68 T (= CGMCC 1.13609 T = JCM 32953 T ) and was isolated from salted brown alga Laminaria produced at Rongcheng, Shandong Province, China.

Declarations Figures
Maximum
-Likelihood phylogenetic tree reconstructions based on 16S rRNA gene (a), rpoB′ gene (b) sequences and 728 conserved single-copy protein sequences (c), showing the relationships between strains HD8-83T, LYG-36T, DLLS-82, RC-68T and related members within the family Halobacteriaceae.Bootstrap values (%) are based on 1000 replicates and are shown for branches with more than 70 % of bootstrap support.Filled circles indicate branches that were recovered in the neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees.Empty circles indicate branches that were recovered in the maximum-likelihood and maximum-parsimony phylogenetic trees.Bar represents expected substitutions per nucleotide position.
Anaerobic growth is observed in the presence of nitrate, DMSO and L-arginine.Indole formation and H 2 S formation are positive.The type strain cannot hydrolyze gelatin, hydrolyze casein, starch or Tween 80.The following substrates are utilized as single carbon and energy sources for growth: D-glucose, D-mannose, D-galactose