The genus Gymnodinialimonas, within the family Paracoccaceae of the phylum Pseudomonadota, was first proposed by Jiang et al. (2022), with Gymnodinialimonas ceratoperidinii J12C1-MA-4^T designated as the type species. At the time of writing (October 2024), the genus includes two valid published species (https://lpsn.dsmz.de/genus/gymnodinialimonas), both isolated from marine dinoflagellates, Ceratoperidinium margalefii and Karlodinium veneficum (Jiang et al., 2022; Peng et al., 2023). Recently, a strain of the genus Gymnodinialimonas isolated from intertidal sediments demonstrated the capability to degrade 4-hydroxybenzoate, a preservative compound, indicating potential for the biodegradation of marine pollutants (Li et al., 2024). Members of the genus Gymnodinialimonas are Gram-stain-negative, aerobic or facultatively anaerobic, rod-shaped, non-motile, and positive for oxidase and catalase activities. They contain ubiquinone (Q)-10 as the major respiratory quinone and summed feature 8 (C_18:1 ω7c and/or C_18:1 ω6c) as the major cellular fatty acid. The major polar lipids include phosphatidylglycerol (PG), phosphatidylcholine (PC), unidentified phospholipid (PL), unidentified aminolipid (AL), and unidentified lipid (L) (Jiang et al., 2022; Peng et al., 2023).

This study isolated strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T as part of an investigation on the bacterial diversity in sponges, mussels, and algae. The objective was to determine their taxonomic position through a polyphasic approach that integrates phylogenetic, genomic, phenotypic, physiological, and chemotaxonomic analysis.

Isolation and ecology

Strain 202GB13-11^T was isolated from the marine sponge, Hymeniacidon sinapium, collected at the coast of Geobukseom, Busan, Republic of Korea (35°04'32.3" N 129°01'20.1" E) in 2020. Strains 2305UL16-5^T and 2307UL20-7^T were isolated from the mussel, Mytilus unguiculatus, and the green algae, Ulva prolifera, respectively, were collected at the coast of Ulleungdo, Gyeongsangbuk-do, Republic of Korea (37°31'07" N 130°47'41" E and 37°30'50" N 130°47'40" E, respectively) in 2023. All samples were ground using a sterilized mortar and pestle, suspended in artificial seawater (ASW; 23.48 g NaCl, 10.64 g MgCl₂∙6H₂O, 3.92 g Na₂SO₄, 1.102 g CaCl₂, and 0.664 g KCl per liter), and serially diluted from 10^-1–10^-4 in ASW. Aliquots (100 µl) were spread onto marine agar 2216 (MA; BD) and incubated at 28°C for 7 days. Colonies were selected, purified by subculturing on MA, and routinely cultivated aerobically on MA at 28°C for 3 days. Strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T were preserved in marine broth 2216 (MB; BD) with 20% (v/v) glycerol at -80°C. To further compare phenotypic and physiological characteristics and analyze fatty acid composition, G. ceratoperidinii KCTC 82770^T, G. phycosphaerae KCTC 82362^T were obtained from the Korean Collection for Type Cultures.

16S rRNA gene sequencing phylogenetic analysis

The 16S rRNA genes of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T were PCR-amplified using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1542R (5'-AGAAAGGAGGTGATCCAG CC-3') (Weisburg et al., 1991). The PCR products were purified using the HiGene^TM Gel & PCR Purification System (BIOFACT, Korea) and sequenced with primers 27F, 785F (5'-GGATTAGATACCCTGGTA-3'), 907R (5'-CCGTCAATTCMTTTRAGTTT-3'), and 1492R (5'-GGTTACCTTGTTACGACTT-3') by Macrogen (Korea). The partial 16S rRNA gene sequences were assembled using SeqMan software to obtain nearly complete 16S rRNA gene sequences. The 16S rRNA gene sequence similarity of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T was calculated with those of all validly published species using EzBioCloud (www.ezbiocloud.net) server. The 16S rRNA gene sequences of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T and closely related type strains were aligned using ClustalW (Thompson et al., 2003) in MEGA11 (Tamura et al., 2021). Phylogenetic trees were constructed using MEGA11 software with the neighbor-joining (NJ), maximum-likelihood (ML), and maximum-parsimony (MP) algorithms, and bootstrap values derived from 1,000 replications. Evolutionary distances were calculated using Kimura’s two-parameter model with complete deletion of gaps for the NJ and ML trees, and the MP tree was reconstructed based on the Subtree-Pruning-Regrafting heuristic method with pairwise deletion of gaps.

Genome features and genome-based phylogeny

Genomic DNA of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T was extracted using the Prokaryote/Eukaryote SEV DNA Purification Kit (Maxwell®) and sequenced on the Illumina HiSeq X platform by Macrogen (Korea). High-quality sequencing reads were de novo assembled using SPAdes version 3.15.5 (Li et al., 2010). The assembled genome sequences were submitted to the GenBank and annotated using the NCBI Prokaryotic Genomes Annotation Pipeline (Tatusova et al., 2016). Genome quality, including completeness and contamination rates was assessed using CheckM (version 1.2.2) on NCBI datasets (Parks et al., 2015). A phylogenetic tree based on concatenated amino acid sequences of 400 marker proteins was constructed using PhyloPhlAn (version 3.1.68) (Segata et al., 2013) using the ML algorithm with bootstrap analyses (1000 replications), and visualized using MEGA 11. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T and reference strains G. ceratoperidinii J12C1-MA-4^T and G. phycosphaerae N5^T were calculated using OrthoANI (Yoon et al., 2017) and the Genome-to-Genome Distance Calculator (http://ggdc.dsmz.de/ggdc.php) (Meier-Kolthoff et al., 2013), respectively. Gene functions and metabolic pathways were annotated using the KEGG Automatic Annotation Server (KAAS, www.genome.jp/kegg/kaas/) (Moriya et al., 2007).

Phenotypic and physiological properties

Gram staining was performed using the Gram stain kit (BD) according to the manufacturer’s instructions. Cell morphology, including shape, size, and flagella presence was examined using a transmission electron microscope (Libra 20, Carl Zeiss) after culturing cells on MA at optimal growth temperature of strains for 3 days. Cell motility was assessed on semi-solid MA in test tubes containing 0.3% agar. Growth of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T was evaluated at different temperatures (4, 10, 15, 20, 22, 25, 28, 30, 32, 35, 40, and 45°C) and pH values (4.0–10.0 1.0 pH unit intervals) for 3 days on MA and MB, respectively. MB with pH ranges of 4.0–5.5, 6.0–8.0, and 8.5–10.0 were prepared using acetate, phosphate, and Tris-HCl buffers, respectively. Growth of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T was tested in ZoBell broth (5 g peptone, 1 g yeast extract, 5.94 g MgSO₄∙7H₂O, 4.53 g MgCl₂∙6H₂O, 0.64 g KCl, 0.01 g FePO₄∙4H₂O, and 1.3 g CaCl₂ per a liter of distilled water, pH 7.6) containing different NaCl concentrations (0, 0.5, 1–10% w/v; at 1% intervals) and incubated at the optimal growth temperature for 3 days. Anaerobic growth was assessed on MA using the GasPak^TM EZ Anaerobe Pouch System (BD) at the optimal growth temperature for 14 days. Hydrolysis of starch, casein, tyrosine, Tween 20, and Tween 80 was tested on MA supplemented with 1% of each substrate. Catalase activity was determined by bubble formation upon exposure of cells to 3% (v/v) H₂O₂, and oxidase activity was assessed using a 1% (w/v) tetramethyl-p-phenylenediamine reagent (Sigma-Aldrich). Enzymatic and biochemical characteristics were determined using API 20NE, API 50CH and API ZYM kits (bioMérieux) after 3 days and 6 h of incubation, respectively, at the optimal growth temperature.

Chemotaxonomy

To determine respiratory quinone, strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T were cultured in MB at their optimal growth temperature until reaching the exponential growth phase (OD₆₀₀ 0.6–0.8). Cells were harvested, and respiratory quinones were extracted using chloroform-methanol (2:1, v/v), dried under vacuum, and re-extracted in n-hexane-water (1:1, v/v). The crude n-hexane-quinone solution was purified using Sep-Pak Silica Vac cartridges (Waters) and analyzed by reverse-phase high-performance liquid chromatography, as described previously (Hiraishi et al., 1996). Polar lipids of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T were extracted from cells harvested during the exponential growth phase following the method of Minnikin et al. (1984) and analyzed by two-dimensional thin-layer chromatography (TLC) using different development solvents: chloroform-methanol-water ratios of 65:25:4 (v/v/v) and chloroform-acetic acid-methanol-water ratios of 80:15:12:4 (v/v/v/v). Specific lipids were detected using 10% ethanolic molybdatophosphoric acid for total polar lipids, ninhydrin for aminolipids, molybdenum blue spray reagent for phospholipids, and Dragendorff reagent for choline. Cellular fatty acids of strains 202GB13-11^T, 2305UL16-5^T, 2307UL20-7^T, and G. phycosphaerae KCTC 82362^T were analyzed by culturing cells on MA for 3 days. Cellular fatty acids were saponified, methylated, and extracted following the Sherlock Microbial Identification System (MIDI) protocol. Fatty acid methyl esters were analyzed using gas chromatography (Agilent 7890A) and identified using MIDI version 6.3 and the TSBA6 database (Miller and Berger, 1985).

16S rRNA gene sequencing phylogenetic analysis

The 16S rRNA gene sequence similarities between strains 202GB13-11^T (1,427 bp), 2305UL16-5^T (1,430 bp), and 2307UL20-7^T (1,428 bp) ranged from 97.1–97.5%. In a comparison of 16S rRNA gene sequences with type strains of the genus Gymnodinialimonas, strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T were most closely related to G. ceratoperidinii J12C1-MA-4^T with 97.3%, 97.0%, and 96.2% similarity, respectively. The strains were also closely related to G. phycosphaerae N5^T with similarities of 96.9%, 97.0%, and 96.1%, respectively. Phylogenetic analysis using the NJ algorithm revealed that strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T formed a monophyletic group with members of the genus Gymnodinialimonas (Fig. 1), while clustering into a distinct clade, indicating their close phylogenetic relationship. Phylogenetic trees reconstructed with the ML and MP algorithms also supported their affiliation within the genus Gymnodinialimonas (Supplementary data Fig. S1). Comparative and phylogenetic analyses based on 16S rRNA gene sequences strongly suggested that strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T represent novel species within the genus Gymnodinialimonas.

Fig. 1. Neighbor-joining tree based on 16S rRNA gene sequences, showing the phylogenetic relationships of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T and closely related taxa. Bootstrap values (> 70%) based on 1,000 replications are shown at branch nodes. Filled circles indicate the corresponding nodes that the relationships were also recovered by maximum-likelihood and maximum-parsimony algorithms. Harenicola maris KMM 3653^T (LC595119) was used as the outgroup. The scale bar of 0.01 changes per nucleotide position.

Genome features and genome-based phylogeny

The whole genome sequence of strain 202GB13-11^T comprised 2 contigs with a total length of 4.0 Mb and an N50 value of 3990.7 kb, strain 2305UL16-5^T comprised 17 contigs with a total length of 4.8 Mb and N50 value of 963.5 kb, and strain 2307UL20-7^T comprised 10 contigs with a total length of 3.8 Mb and an N50 value of 2021.6 kb. The DNA G + C contents of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T were 60.8%, 60.6%, and 63.0%, respectively. Genome completeness and contamination rates, assessed by CheckM, were 99.7%, 99.3%, and 100.0% and 0%, 0.8%, and 0%, respectively, indicating that the de novo assembled genome has high quality. The 16S rRNA gene sequences of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T obtained from PCR were identical to those from their whole-genome sequences. General genomic features, such as genome size and DNA G + C content, were consistent with members of the genus Gymnodinialimonas (Table 1). ANI and dDDH values between strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T and the reference strains ranged from 70.7–79.6% and 13.0–35.7%, respectively (Table 1). Both values are below the species delineation thresholds of 95–96% ANI and 70% dDDH (Richter and Rosselló-Móra, 2009). Phylogenomic analysis based on concatenated amino acid sequences of 400 marker proteins showed that strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T formed a clade within the genus Gymnodinialimonas, consistent with the results of 16S rRNA gene sequence-based analysis (Fig. 2). These results indicated that strains 202GB13-11^T, 2305 UL16-5^T, and 2307UL20-7^T represent novel species within the genus Gymnodinialimonas.

Table 1

General genomic features of strains 202GB13-11^T, 2305UL16-5^T, 2307UL20-7^T and closely related type strains

Strains: 1, 202GB13-11^T; 2, 2305UL16-5^T; 3, 2307UL20-7^T; 4, G. ceratoperidinii J12C1-MA-4T; 5, G. phycosphaerae N5^T.

Feature		1	2	3	4	5
Genome size (Mb)		4.0	4.8	3.8	3.6	4.3
Genome status		Draft	Draft	Draft	Complete	Complete
No. of contigs		2	17	10	1	2
N50 value (kb)		3990.7	963.5	2021.5	3621.9	4308.0
G + C content (%)†		60.8	60.6	63.0	64.5	62.9
No. of genes		4101	4732	3802	3494	4329
No. of protein coding genes (CDS)		4040	4683	3754	3446	4281
No. of pseudogenes		14	24	10	18	51
No. of tRNA genes		41	43	42	42	42
GenBank accession number		JBFRBH000000000	JBBIFW000000000	JBBIFX000000000	CP079194	JAIMBW000000000
ANI value (%)		DDH value (%)
	1		16.8	35.7	18.6	19.9
	2	75.5		17.4	15.2	15.7
	3	74.7	76.1		18.7	20.1
	4	74.7	76.1	79.2		30.8
	5	70.7	70.9	71.0	71.0

^†DNA G + C content were calculated from whole genome sequence

Fig. 2. Maximum-likelihood tree based on concatenated amino acid sequences of 400 marker proteins showing the phylogenetic relationships between strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T, and related taxa. Harenicola maris KMM 3653^T (JADQAZ000000000) was used as the outgroup. Bootstrap values (> 70%) based on 1,000 replications are shown at branch nodes. The scale bar of 0.1 changes per amino acid position.

Bacteria are known to establish symbiotic relationships with various marine organisms, such as sponges, mussels, and, algae, enhancing the growth, survival, and reproduction of their host by producing and supplying essential B vitamins (Sañudo-Wilhelmy et al., 2014; Wang et al., 2024). Genome analysis of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T using KEGG functional annotation revealed that all three strains harbor genes for the biosynthesis of riboflavin (B₂) from ribulose 5-phosphate (ribBEH) and cobalamin from glycine (5-aminolevulinate synthase gene, hemBCD, and cobABCFGHIJKLMNPQSTV). However, only strain 2305UL16-5^T was found to biosynthesize thiamine from 5-aminoimidazole ribotide (thiCDEN and thiamine phosphate phosphatase gene). These biosynthetic potential suggest that strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T may contribute to the growth and survival of their host organisms.

Marine organisms are often exposed to oxidative stress due to various environmental factors, and bacteria mitigate oxidative damage and maintain cellular homeostasis by synthesizing glutathione and ophthalmate through specific biosynthetic pathways (Hellou et al., 2012; Musgrave et al., 2013; Narainsamy et al., 2016). Strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T harbor genes for glutathione synthesis from L-glutamate (gshAB) and ophthalmate synthesis from L-serine (glyA, ltaE, CHA1 , ilvE, and gshAB), suggesting that these strains could play a key role in protecting their host organisms from oxidative stress.

Phenotypic and physiological properties

Cells of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T were Gram-stain-negative, non-motile, rod-shaped, oxidase- and catalase-positive. Cells of strain 202GB13-11^T measured 0.8–1.0 µm in width and 1.0–1.2 µm in length, strain 2305UL16-5^T cells were 0.7–1.1 µm in width and 1.0–1.8 µm in length, and strain 2307UL20-7^T cells were 0.6–0.8 µm in width and 0.9–1.6 µm in length (Supplementary data Fig. S2). Colonies of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T were circular, smooth, and convex with entire margins, and their colony colors were pale yellow-, light pink-, and pale yellow-colored, respectively, after 3 days of incubation on MA. Strains 202GB13-11^T and 2305UL16-5^T did not exhibit anaerobic growth after 14 days of anaerobic incubation on MA at 28°C, whereas strain 2307UL20-7^T showed weak anaerobic growth. Phenotypic properties of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T, are summarized in Table 2 and Supplementary data Table S1. However, some other phenotypic properties, such as nitrate reduction, glucose fermentation, hydrolysis of tyrosine, Tween 80, and gelatin, acid production from D-arabinose, L-arabinose, ribose, L-xylose, galactose, fructose, mannose, maltose, D-tagatose, D-arabitol, potassium 5-keto-gluconate, and activities of lipase (C14), cystine arylamidase, naphthol- AS-BI-phosphohydrolase, β-galactosidase, β-glucosidase, and N -acetyl-β-glucosaminidase, differ from their closest relatives within the genus Gymnodinialimonas. These differences in enzymatic activities and metabolic properties highlight the distinct biochemical profiles of the novel strains, further supporting their classification as separate species within the genus.

Table 2

Comparisons of phenotypic, physiological, and chemotaxonomic characteristics of strains 202GB13-11^T, 2305UL16-5^T, 2307UL20-7^T and closely related type strains

Strains: 1, 202GB13-11^T (this study); 2, 2305UL16-5^T (this study); 3, 2307UL20-7^T (this study); 4, G. ceratoperidinii KCTC 82770^T (Jiang et al., 2022); 5, G. phycosphaerae KCTC 82362^T (Peng et al., 2023). All strains are positive for hydrolysis of aesculin and activity of oxidase, catalase, valine arylamidase, β-galactosidase (4-nitrophenyl-βD-galactopyranoside), alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, and acid phosphatase. All strains are negative for motility, Gram staining, indole production, activity of arginine dihydrolase, trypsin, β-galactosidase, and α-fucosidase, and assimilation of L-arabinose, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Symbols: +, positive; w, weakly positive; –, negative.

Characteristic	1	2	3	4	5
Isolation source	Sponge	Mussel	Seaweed	Dinoflagellate	Dinoflagellate
Colony color	pale yellow	light pink	pale yellow	pale yellow	pale yellow
Nitrate reduction to nitrite*	–	–	+	–	+
Glucose fermentation*	–	–	+	+	–
Growth range (optimum):
Temperature (℃)	4–40 (30)	4–35 (28)	4–40 (28)	4–35 (30)	10–35 (25)
pH	6.0–9.0 (7)	6.0–10.0 (7)	6.0–10.0 (8)	5.0–8.0 (7)	5.0–8.0 (7)
NaCl (%, w/v)	0–8.0 (2)	0.5–7.0 (2–3)	0.5–7.0 (2)	0–8.0 (1–2)	0–3.0 (1)
Hydrolysis* of:
Starch, casein, Tween 20	–	–	–	–	–
Tyrosine	+	–	–	–	–
Tween 80	–	+	–	–	–
Gelatin	+	+	–	–	–
Assimilation* of:
D-Glucose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate	–	–	+	–	–
Enzyme activity* of:
Urease	+	+	+	–	–
Lipase (C14)	–	–	w	–	–
Cystine arylamidase	–	w	w	w	w
α-Chymotrypsin	–	–	–	w	–
Naphthol-AS-BI-phosphohydrolase	w	–	–	–	w
α-Galactosidase	–	–	–	w	–
β-Glucuronidase	w	w	–	w	–
α-Glucosidase	+	w	w	+	–
β-Glucosidase	+	–	w	+	–
α-Mannosidase	–	–	–	+	–
N-Acetyl-β-glucosaminidase	–	+	–	–	–
Polar lipids†	DPG, PG, PC, AL, 2PL, 3L	PG, PC, 3AL, 2PL, L	PG, PC, 2AL, 2PL, 2L	PG, PC, PL, AL, L	PG, PE, PC

* These analyses were conducted under the same conditions in this study

^† DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; AL, unidentified aminolipid; PL, unidentified phospholipid; L, unidentified lipid

Chemotaxonomy

Q-10 was detected as the sole respiratory quinone in strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T, consistent with other members of the genus Gymnodinialimonas. These strains exhibited polar lipids profile, including phosphatidylglycerol (PG), phosphatidylcholine (PC), unidentified phospholipid (PL), unidentified aminolipid (AL), and unidentified lipid (L) (Supplementary data Fig. S3). However, diphosphatidylglycerol (DPG) was identified only in strain 202GB13-11^T. The identification of PG and PC as dominant polar lipids aligned with those found in G. ceratoperidinii KCTC 82770^T and G. phycosphaerae N5^T (Jiang et al., 2022; Peng et al., 2023). The predominant fatty acids in strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T were summed feature 8 (comprising C_18:1 ω6c and/or C_18:1 ω7c). Additionally, strain 2307UL20-7^T was found to have more C_16:0 than other strains. Although their fatty acid profiles were generally consistent with those of the G. ceratoperidinii J12C1-MA-4^T and G. phycosphaerae KCTC 82362^T, notable variations were observed in components such as C_19:0 cyclo ω8c and summed feature 7 (comprising C_19:1 ω6c and/or C_19:1 ω7c) (Table 3).

Table 3

Comparison of the cellular fatty acid compositions (%) of strains 202GB13-11^T, 2305UL16-5^T, 2307UL20-7^T and closely related type strains

Strains: 1, 202GB13-11^T (this study); 2, 2305UL16-5^T (this study); 3, 2307UL20-7^T (this study); 4, G. ceratoperidinii J12C1-MA-4^T; 5, G. phycosphaerae KCTC 82362^T. Major components (>10.0%) are highlighted in bold. ND, no data available; tr, trace amount (<0.5); –, no detected.

Fatty acids	1	2	3	4a	5
Saturated
C14:0	tr	0.5	0.5	–	1.0
C16:0	7.9	9.4	15.0	4.1	5.3
C18:0	1.9	1.0	1.6	1.2	1.9
Unsaturated
C18:1 ω7c 11-methyl	3.4	5.2	6.6	3.8	–
C19:0 cyclo ω8c	2.0	–	–	ND	1.1
C20:1 ω7c	tr	tr	tr	–	0.9
Hydroxy
C10:0 3OH	3.1	2.2	2.6	2.0	2.5
C16:0 2OH	9.9	6.5	1.4	3.5	7.2
C18:1 2OH	3.6	4.6	0.5	3.7	–
Summed feature*
3	1.2	0.5	1.0	1.1	1.4
7	–	tr	–	ND	0.7
8	65.0	68.9	70.0	79.1	64.6

^a Data from Jiang et al. (2022)

* 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 comprised C_16:1 ω6c and/or C_16:1 ω7c; summed feature 7 comprised C_19:1 ω6c and/or C_19:1 ω7c; summed feature 8 comprised C_18:1 ω7c and/or C_18:1 ω6c.

Taxonomic conclusion

Phylogenetic analysis of the 16S rRNA gene and whole-genome sequences confirmed that strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T belong to the genus Gymnodinialimonas. Further evidence, including 16S rRNA gene sequence similarities, genome relatedness including ANI and digital DDH values, and distinct phenotypic and physiological characteristics–such as the ability to hydrolyze tyrosine, Tween 80, and gelatin, enzyme activity of urease, lipase (C14), and N -acetyl-β-glucosaminidase, and polar lipids profile–supports their classification as novel species of the genus Gymnodinialimonas. The proposed names for these species are Gymnodinialimonas hymeniacidonis sp. nov., Gymnodinialimonas mytili sp. nov., and Gymnodinialimonas ulvae sp. nov.

Description of Gymnodinialimonas hymeniacidonis sp. nov.

Gymnodinialimonas hymeniacidonis (hy.me.ni.a.ci.do’nis. N.L. gen. fem. n. hymeniacidonis, of the sponge Hymeniacidon sinapium).

Cells are Gram-stain-negative, aerobic, and non-motile short rods lacking flagella (0.8–1.0 µm in width and 1.0–1.2 µm in length). Colonies are pale yellow-colored, circular, smooth, and convex with entire margins after incubation at 30℃ for 3 days. Growth occurs at 4–40℃ (optimum, 30℃), pH 6.0–9.0 (optimum, pH 7.0), and 0–8.0% (w/v) NaCl (optimum, 2.0%). Oxidase- and catalase-positive. Indole production and glucose fermentation are negative. Nitrate is not reduced to nitrite. Aesculin, tyrosine, and gelatin are hydrolyzed, whereas starch, casein, Tween 20, and Tween 80 are not. All assimilation results for API 20NE are negative. Acid is produced from ribose, L-xylose, fructose, esculin ferric citrate, D-tagatose, D-arabitol, and potassium 5-keto-gluconate, but not from the other substrates. Valine arylamidase, β-galactosidase (4-nitrophenyl-βD-galactopyranoside), alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, α-glucosidase, urease, Naphthol-AS-BI-phosphohydrolase, β-glucuronidase, and β-glucosidase activities are positive, but arginine dihydrolase, trypsin, β-galactosidase, α-mannosidase, α-fucosidase, lipase (C14), cystine arylamidase, α-chymotrypsin, α-galactosidase, and N -acetyl-β-glucosaminidase activities are negative. Q-10 is the sole respiratory quinone, and the major polar lipids are DPG, PG, PC, two unidentified PLs, an unidentified AL, and three unidentified Ls. The major cellular fatty acids are summed feature 8 (comprising C_18:1 ω6c and/or C_18:1 ω7c).

The type strain, 202GB13-11^T (=KACC 23695^T=JCM 37088^T), was isolated from a marine sponge, Hymeniacidon sinapium, collected from the coast of Geobukseom, Busan, Republic of Korea. The DNA G + C content is 60.8%. The GenBank accession numbers for the 16S rRNA gene and genome sequences are PP627012 and JBFRBH000000000, respectively.

Description of Gymnodinialimonas mytili sp. nov.

Gymnodinialimonas mytili (my’ti.li. N.L. gen. masc. n. mytili, of Mytilus, named after the generic name of the mussel Mytilus unguiculatus, from which the type strain was isolated).

Cells are Gram-stain-negative, aerobic, and non-motile short rods lacking flagella (1.7–1.1 µm in width and 1.0–1.8 µm in length). Colonies are light pink-colored, circular, smooth, and convex with entire margins after incubation at 28℃ for 3 days. Growth occurs at 4–35℃ (optimum, 28℃), pH 6.0–10.0 (optimum, pH 7.0), and 0.5–7.0% (w/v) NaCl (optimum, 2.0–3.0%). Oxidase- and catalase-positive. Indole production and glucose fermentation are negative. Nitrate is not reduced to nitrite. Aesculin, Tween 80, and gelatin are hydrolyzed, whereas tyrosine, starch, casein, and Tween 20 are not. All assimilation results for API 20NE are negative. Acid is produced from D-arabinose, ribose, esculin ferric citrate, maltose, D-tagatose, and potassium 5-keto-gluconate, but not from the other substrates. Valine arylamidase, β-galactosidase (4-nitrophenyl-βD-galactopyranoside), alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, α-glucosidase, urease, cystine arylamidase, β-glucuronidase, and N -acetyl-β-glucosaminidase activities are positive, but arginine dihydrolase, trypsin, β-galactosidase, α-mannosidase, α-fucosidase, lipase (C14), α-chymotrypsin, Naphthol-AS-BI-phosphohydrolase, α-galactosidase, and β-glucosidase are negative. Q-10 is the sole respiratory quinone, and the major polar lipids are PG, PC, two unidentified PLs, three unidentified ALs, and an unidentified L. The major cellular fatty acids are summed feature 8 (comprising C_18:1 ω6c and/or C_18:1 ω7c).

The type strain, 2305UL16-5^T (= KACC 23693^T = JCM 37091^T), is isolated from a mussel, Mytilus unguiculatus, collected from Ulleungdo, Republic of Korea. The DNA G + C content is 60.6%. The GenBank accession numbers for the 16S rRNA gene and genome sequences are PP627013 and JBBIFW000000000, respectively.

Description of Gymnodinialimonas ulvae sp. nov.

Gymnodinialimonas ulvae (ul’vae. L. gen. fem. n. ulvae, of Ulva, the name of the seaweed species from which is isolated).

Cells are Gram-stain-negative, facultative anaerobic, and non-motile rods lacking flagella (0.6–0.8 µm in width and 0.9–1.6 µm in length). Colonies are pale yellow-colored, circular, smooth, and convex with entire margins after incubation at 28℃ for 3 days. Growth occurs at 4–40℃ (optimum, 28℃), pH 6.0–10.0 (optimum, pH 8.0), and 0.5–7.0% (w/v) NaCl (optimum, 2.0–3.0%). Oxidase- and catalase-positive. Nitrate is reduced to nitrite. Production of indole is negative. Glucose fermentation is positive. Aesculin is hydrolyzed, whereas tyrosine, gelatin, starch, casein, tween 20, and tween 80 are not. Assimilation of D-glucose, D-mannose, D-mannitol, N -acetyl-glucosamine, D-maltose, and potassium gluconate are positive, but L-arabinose, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid are negative. Acid is produced from L-arabinose, ribose, L-xylose, galactose, mannose, esculin ferric citrate, D-tagatose, D-arabitol, and potassium 5-keto-gluconate, but not from the other substrates. Valine arylamidase, β-galactosidase (4-nitrophenyl-βD-galactopyranoside), alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, α-glucosidase, urease, lipase (C14), cystine arylamidase, and β-glucosidase activities are positive, but arginine dihydrolase, trypsin, β-glucuronidase, α-mannosidase, α-fucosidase, α-chymotrypsin, Naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, and N -acetyl-β-glucosaminidase activities are negative. Q-10 is the sole respiratory quinone, and the major polar lipids are PG, PC, two unidentified PLs, two unidentified ALs, and two unidentified Ls. The major cellular fatty acids are summed feature 8 (comprising C_18:1 ω6c and/or C_18:1 ω7c) and C_16:0.

The type strain, 2307UL20-7^T (=KACC 23694^T=MCCC 1K09527^T), was isolated from green algae, Ulva prolifera, collected from the coast of Ulleungdo, Gyeongsangbuk-do, Republic of Korea. The DNA G + C content is 63.0%. The GenBank accession numbers for the 16S rRNA gene and genome sequences are PP627014 and JBBIFX000000000, respectively.

Emended description of Gymnodinialimonas phycosphaerae Peng et al. 2023

The species description is as given for Gymnodinialimonas phycosphaerae (Peng et al., 2023) with the following amendments. The major cellular fatty acids are summed feature 8 (comprising C_18:1 ω6c and/or C_18:1 ω7c).

The type strain is N5^T (=KCTC 82362^T=NBRC 114899^T). The GenBank accession numbers for the 16S rRNA gene and genome sequences of the type strain are MZ329063 and JAIMBW000000000, respectively.

그람 음성, 비운동성, 막대 모양의 세균인 202GB13-11^T, 2305UL16-5^T, 2307UL20-7^T은 대한민국 부산과 울릉도 연안에서 채집된 해양 생물(해면, 홍합, 조류)에서 분리되었다. 16S rRNA 유전자 염기서열을 기반으로 한 계통학적 분석 결과, 이 균주들은 Gymnodinialimonas 속 내에서 단일 계통군을 형성하며, Gymnodinialimonas ceratoperidinii J12C1-MA-4^T와 각각 97.3%, 97.0%, 96.2%로 가장 높은 유사성을 나타냈다. 이들 균주는 28–30°C, 2–3% NaCl, pH 7–8에서 최적 생장이 관찰되었다. 이들 균주에서는 공통적으로 유비퀴논-10이 주요 호흡 퀴논으로, summed feature 8 (C_18:1 ω6c 및/또는 C_18:1 ω7c 포함)이 주요 세포 지방산이었다. 주요 극성 지질은 포스파티딜글리세롤, 포스파티딜에탄올아민, 미확인 인지질, 미확인 아미노지질, 미확인 지질로 구성되었다. 202GB13-11^T, 2305UL16-5^T, 2307UL20-7^T 균주들의 DNA G + C 함량은 각각 60.8%, 60.6%, 63.0%였다. 이들 균주와 기준 균주 간의 ANI와 디지털 DNA-DNA 혼성화 값은 각각 70.7–79.6% 및 13.0–35.7% 범위였다. 계통학적, 유전체적, 화학분류학적 특성에 기반하여, 202GB13-11^T, 2305UL16-5^T, 2307UL20-7^T 균주들을 Gymnodinialimonas 속에 속하는 세 개의 신종으로 나타나며, 종명은 Gymnodinialimonas hymeniacidonis sp. nov., (202GB13-11^T = KACC 23695^T = JCM 37088^T), Gymnodinialimonas mytili sp. nov., (2305UL16-5^T=KACC 23693^T = JCM 37091^T), Gymnodinialimonas ulvae sp. nov., (2307UL20-7^T = KACC 23694^T=MCCC 1K09527^T)로 제안한다.

This research was supported by the management of Marine Fishery Bio-resources Center (2024) funded by the National Marine Biodiversity Institute of Korea (MABIK), and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1I1A3046479).

The authors declare no competing financial conflicts of interests.

JYS and JSP conceived and designed the study. JYS and KHK carried out the experimental work, performed data analysis, and prepared the initial draft of the manuscript. Both JYS and KHK contributed to revising and refining the manuscript. JSP provided oversight and supervision throughout the project, guiding its direction and ensuring scientific accuracy.

The GenBank accession numbers for the 16S rRNA gene sequences and genome sequence of strains 202GB13-11^T, 2305UL16-5^T, and 2307UL20-7^T were PP627012, PP627013, and PP627014, and JBFRBH000000000, JBBIFW000000000, and JBBIFX000000000, respectively.