Katagnymene terrestris sp. nov. (Gomontiellaceae, Cyanobacteria) Isolated from the Soil between Rocks in the Republic of Korea

: Soil cyanobacterium, FBCC-A195 were isolated from the soil underneath the Jangnak Bridge, Republic of Korea. The FBCC-A195 was studied using light microscopy (LM), transmission electron microscopy (TEM), 16S rRNA, 16S–23S ITS, and ecological data. FBCC-A195 showed the circular cross-section of the trichome, broad mucilaginous envelopes, a swirl-like pattern of thylakoids inside the cell, and the lowest length/width ratio within the genus. In the 16S rRNA phylogeny, FBCC-A195 showed a sister relationship of Hormoscilla and belonged to the family Gomontiellaceae. The p -distance of FBCC-A195 ranged from 1.7% with H . pringsheimii to 12% with Komvophoron kgarii in the family. In the secondary structure of 16S–23S ITS, D1–D1’, Box-B, and V3 helix of FBCC-A195 were distinguished from those of other taxa in the Gomontiellaceae. Based upon morphological, ecological, and molecular traits, Katagnymene terrestris proved to be a unique and novel species of the Katagnymene .


Introduction
The family Gomontiellaceae (order Gomontiellales) are filamentous non-heterocystous cyanobacteria. The group has been rarely reported in nature; however, it has been primarily identified based on its distinctive morphological features and is recognized for its worldwide distribution in both terrestrial and aquatic habitats. Morphologically, Gomontiellaceae is classified as a short-cell lineage, characterized by numerous segmentations along the filament. Regarding cell shape, the cross-section of trichomes in each taxon exhibited distinct characteristics, including oval, arcuated, or triangular forms, which were then used to classify the respective genera [1][2][3]. Thylakoids are irregularly dispersed throughout the cell and denser in peripheral parts and form swirl-like structures that are demonstrated as a distinctive thylakoid arrangement [1]. According to a monograph of the Oscillatoriales [4], the Gomontiellaceae has included Crinalium by Crow, Gomontiella by Teodoresco, Hormoscilla by Anagnostidis and Komárek, Katagnymene by Lemmermann, Komvophoron by Anagnostidis and Komárek, and Starria by Lang.
The Gomontiellaceae was established based on the Gomontiella [5]. The subfamily Hormoscilloideae was established on the basis of the Hormoscilla, then included Katagnymene with cylindrical to circular cells in cross-section and both genera habitats in the seawater and freshwater [6]. Recently, the genus Komvophoron was added as a member of the Gomontiellaceae based on features of mucus secretion and irregular thylakoid arrangement [3,7,8]. However, the family Gomontiellaceae lacks reliable molecular sequence

Sample Collections and Cultures
A natural soil sample was collected from underneath the Jangnak Bridge located at Ha-Dong, Youngtong-Gu, Suwon, Gyeonggi-do on 14 December 2018 (37 • 17 21.9 N 127 • 03 41.1 E) (Figure 1). The collection of soil cyanobacteria was conducted through a delicate process involving the gentle scraping of the soil surface using a soft brush or sterilized spatula, with the exception of mosses [14]. After the collected natural sample was kept at 4 • C and transported to the lab, the Algal Culture Collection of Kyonggi University (ACKU).
To obtain unicyanobacterial culture, a single filament was isolated under the light microscope (LM) using a Pasteur's pipette (Hilgenberg GmbH, Mansfeld, Germany) and placed in the 24-well plate (SPL, Pocheon, Republic of Korea) with BG-11 liquid medium [15]. After one to two weeks, the unialgal-cultured trichomes were transported to a 50 mL cell culture flask (SPL, Pocheon, Republic of Korea) for mass culture [16]. The synthetic culture was conducted under the temperature of 20-25 • C, the photoperiod of 16 h:8 h (light:dark), the illumination of 25 µmol photons·m −2 ·s −1 . A diluted oligotroph of 1 × 10 −1 BG-11 medium was used to observe trichomes, a variety of cells, and mucilage in the process of mass culture.
Reference strain for species has been deposited at Freshwater Bioresources Culture Collection (FBCC, https://fbp.nnibr.re.kr/fbcc/, accessed on 23 September 2019) at the Nakdonggang National Institute of Biological Resources (NNIBR) of the Republic of Korea a with an accession number of FBCC-A195. The inoculated subculture of the reference strain (FBCC-A195) was preserved in 4% (v/v) formaldehyde and deposited in the Herbarium at the National Institute of Biological Resources (KB, https://species.nibr.go.kr/, accessed on 12 April 2021) (culture aliquot NIBRCY0000001277).

Morphological Analysis and Characterization
For the purpose of facilitating a more detailed observation of natural morphology, soil samples collected underwent hydration using third-distilled water. Subsequently, the hydrated samples were observed and photographed (Olympus UC-90, Olympus, Tokyo, Japan) under a light microscope (Olympus BX53, Olympus, Tokyo, Japan) at magnifications ranging from 100 to 1000 times. Additionally, to explore diverse morphological characteristics, unicyanobacteria cultivated over two weeks were subjected to staining using Indian ink and an aqueous solution of methylene blue.
The ultrastructure of cell was observed and photographed with the transmission electron microscope (TEM). Samples were fixed with 2% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (pH 7.4) at 4 °C for 1 h and then postfixed with 2% osmium tetroxide and 3% potassium hexacyanoferrate at 4 °C for 40 min. The samples were dehydrated in a graded series of ethanol and embedded into LR white resin. Ultra-thin, 80 nm sample sections were conducted using an ultra-cut microtome (Leica Co., Greenwood Village, CO, USA) and placed on a coated square copper grid. The final samples were stained with uranyl acetate and lead citrate. The TEM images were taken at 120 kV using a field emission electron microscope (JEM-2100F) (Jeol, Tokyo, Japan) coupled with a OneView camera (Gatan, Pleasanton, CA, USA) at the Korean Basic Science Institute (KBSI), Chuncheon [17].
We followed Strunecký et al. [9] for the taxonomic classification of cyanobacteria and were advised of the AlgaeBase [8]. Moreover, we referred to Komárek and Anagnostidis [4] to identify the cyanobacteria.

Morphological Analysis and Characterization
For the purpose of facilitating a more detailed observation of natural morphology, soil samples collected underwent hydration using third-distilled water. Subsequently, the hydrated samples were observed and photographed (Olympus UC-90, Olympus, Tokyo, Japan) under a light microscope (Olympus BX53, Olympus, Tokyo, Japan) at magnifications ranging from 100 to 1000 times. Additionally, to explore diverse morphological characteristics, unicyanobacteria cultivated over two weeks were subjected to staining using Indian ink and an aqueous solution of methylene blue.
The ultrastructure of cell was observed and photographed with the transmission electron microscope (TEM). Samples were fixed with 2% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (pH 7.4) at 4 • C for 1 h and then postfixed with 2% osmium tetroxide and 3% potassium hexacyanoferrate at 4 • C for 40 min. The samples were dehydrated in a graded series of ethanol and embedded into LR white resin. Ultra-thin, 80 nm sample sections were conducted using an ultra-cut microtome (Leica Co., Greenwood Village, CO, USA) and placed on a coated square copper grid. The final samples were stained with uranyl acetate and lead citrate. The TEM images were taken at 120 kV using a field emission electron microscope (JEM-2100F) (Jeol, Tokyo, Japan) coupled with a OneView camera (Gatan, Pleasanton, CA, USA) at the Korean Basic Science Institute (KBSI), Chuncheon [17].
We followed Strunecký et al. [9] for the taxonomic classification of cyanobacteria and were advised of the AlgaeBase [8]. Moreover, we referred to Komárek and Anagnostidis [4] to identify the cyanobacteria.

DNA Extraction, PCR, and Sequencing
Genomic DNA of the strain FBCC-A195 was extracted using the LaboPass Tissue Genomic DNA Isolation Kit Mini (Cosmogenetech, Seoul, Republic of Korea) according to the manufacturer's instructions. PCR and sequencing reactions of 16S-23S rDNA gene were conducted using cyanobacterial specific primer set, i.e., 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and CY-23R600 (5 -CGGCTCATTCTTCAACAGGCAC-3 ) [18]. The PCR amplification was carried out using the Maxime™ i-Stars Taq PCR premix (iNtRON, Daejeon, Republic of Korea) in a total volume of 20 µL including 17 µL of sterile distilled water, 1 µL of each primer (10 pmoles), and 1 µL of template DNA. The reaction consisted of an initial denaturation at 94 • C for 5 min, followed by 35 main cycles of denaturation at 94 • C for 20 s, annealing at 55 • C for 30 s, extension at 72 • C for 90 s, and a final extension at 72 • C for 10 min [18]. Amplified PCR products were purified with the MEGAquick-spin™ Plus Fragment DNA Purification Kit (iNtRON, Daejeon, Republic of Korea) and sent to a commercial Sanger sequencing service (Macrogen, Seoul, Republic of Korea). The electropherogram of both forward and reverse strands was compiled using Geneious Prime (http://www.geneious.com; Biomatters, Auckland, New Zealand) and confirmed manually. All newly determined 16S rRNA and 16S-23S ITS sequences were deposited in GenBank, under accession no. MZ536612 (https://www.ncbi.nlm.nih.gov, accessed on 14 July 2021).

Alignment, Phylogenetic Analyses, and Secondary Structure
Published 16S rRNA gene sequences of Gomontiellaceae (e.g., Mikhailyuk et al. [2]) and oscillatorian cyanobacterium representatives (minimum 584 bp, average 1305 bp, and maximum 1491 bp in length) were obtained from GenBank and aligned using the ClustalW implemented in Geneious Prime before manual editing. Pseudanabaena catenata SAG254 (1464 bp; Synechococcales) and Gloeobacter violaceus PCC 7421 (1353 bp; Gloeobacterales) were used as outgroups. All positions (ambiguous and unambiguous) of alignment were used in subsequence analyses. Total alignment is available from the corresponding author upon request.
The phylogeny and statistical supports for monophyletic nodes were inferred under the Maximum Likelihood (ML) method using RAxML v8.2.12 [19], the Neighbor-Joining (NJ) method using MEGA X 10.2.6 [20], and the Bayesian Inference (BI) using MrBayes v3.2.7a [21]. A general time-reversible model with rate heterogeneity (GTR + G) was applied for the ML and BI. The "f-a" option was used in RAxML for simultaneous best phylogenetic tree search with rapid bootstrap analysis with "-# 1000" (1000 bootstrap replications; MLB), default "-I" (automatically optimized SPR branch rearrangement for heuristic search), and "-c" (25 distinct rate categories). The default p-distance setting for sequence differences was used for NJ tree search and 1000 bootstrap replications (NJB). The p-distances among taxa were calculated in the program MEGA X 10.2.6. Bayesian phylogeny and posterior probabilities inferred from 50 million generations of the Metropolis-coupled Markov Chain Monte Carlo (MC 3 ) with default parameters: two independent runs with different random start points, one cold chain and three heated chains for each run, and tree sampling at every 500th generation. The burn-in point of chain was identified by the average standard deviation of split frequencies (<0.01) between runs, i.e., 12 million generations. Thus, initial 25% generations were discarded for the Bayesian posterior probability (BPP) calculation. The secondary structure of 16S-23S ITS was inferred with a considering Mikhailyuk et al. [2]. Helices were folded with the web-based software Mfold [22] and re-drawn in the PseudoViewer3 [23] for easy comparison with available structures from relative taxa.

Alternative Topology Tests
Alternative family level relationships were evaluated using the approximately unbiased (AU) test [24] implemented in program CONSEL version 0.1k [25]. The 16S rRNA data were used for paired-site tests with the best ML phylogeny and all possible alternative phylogenies among closely related families. The sister relationships of Katagnymene terrestris FBCC-A195 and Trichodesmium pelagicum str. JWI1 were compared (i) within Gomontiellaceae and (ii) within Microcoleaceae as well. Three independent tests were performed with 100,000 bootstrap replicates using the same evolutionary model, i.e., GTR + G used in ML search.

Morphological Characterization
In the natural sample, K. terrestris exhibits a floating appearance, with mucilage accumulation around each individual (Figure 2A,B). In the cultured strain, the trichome of K. terrestris FBCC-A195 exhibits a circular cross-section shape and is surrounded by mucilage, aligning with the characteristic morphologies of Katagnymene (Figure 3). The arrangement of thylakoids in a swirl-like pattern and the presence of pit-like and junctional pores within the cell wall is evident (Figure 4). Using Indian ink and an aqueous solution of methylene blue, it observed the envelopes and confirmed the presence of a mucilaginous separation disk ( Figure 3A,B). Moreover, the presence of mucous microfibrils in the crosscell wall was observed in K. terrestris FBCC-A195 ( Figure 3G). These distinct morphological characteristics unequivocally distinguish K. terrestris FBCC-A195.

Alternative Topology Tests
Alternative family level relationships were evaluated using the approximately unbiased (AU) test [24] implemented in program CONSEL version 0.1k [25]. The 16S rRNA data were used for paired-site tests with the best ML phylogeny and all possible alternative phylogenies among closely related families. The sister relationships of Katagnymene terrestris FBCC-A195 and Trichodesmium pelagicum str. JWI1 were compared (i) within Gomontiellaceae and (ii) within Microcoleaceae as well. Three independent tests were performed with 100,000 bootstrap replicates using the same evolutionary model, i.e., GTR + G used in ML search.

Morphological Characterization
In the natural sample, K. terrestris exhibits a floating appearance, with mucilage accumulation around each individual (Figure 2A,B). In the cultured strain, the trichome of K. terrestris FBCC-A195 exhibits a circular cross-section shape and is surrounded by mucilage, aligning with the characteristic morphologies of Katagnymene ( Figure 3). The arrangement of thylakoids in a swirl-like pattern and the presence of pit-like and junctional pores within the cell wall is evident (Figure 4). Using Indian ink and an aqueous solution of methylene blue, it observed the envelopes and confirmed the presence of a mucilaginous separation disk ( Figure 3A,B). Moreover, the presence of mucous microfibrils in the cross-cell wall was observed in K. terrestris FBCC-A195 ( Figure 3G). These distinct morphological characteristics unequivocally distinguish K. terrestris FBCC-A195.

16 S rRNA Characteristics and Phylogeny
The

Taxonomic Treatment
On the basis of the results of polyphasic analysis, we propose the following descriptions of new cyanobacterial taxa under the provisions of the ICN (International Code of Nomenclature for algae, fungi, and plants) [26].
Order  (Figures 2-4). Description: Solitary filaments or occasional irregular clusters, straight or bent. Mucilage (discernible through methylene blue staining) colorless, non-lamellated, diffuse, dense, attached to or separate from trichomes, broadly enveloping. Broad mucilaginous sheaths (visible with India ink staining), delicate, diffuse, measuring 50.0-53.0 µm in width. Trichomes ranging from blue-green to olive, with non-tapering ends, not or slightly constricted at cross walls, measuring 4.0-14.6 µm in width. Cells shorter than wide, displaying a circular cross-section, measuring 1.35-1.54 µm in length. Apical cells are rounded, truncated, convex, or nearly hemispherical, featuring a slightly thickened cell wall and a calyptra. Thylakoid pattern is either parietal or helically twisted tangles, exhibiting a distinct arrangement. Reproduction involves trichome disintegration into immotile, often elongated hormocytes through specialized separation discs, remaining within the mucilage, or occasional fragmentation through necrotic cells.
Diagnosis: Katagnymene distinguishes itself from Hormoscilla in terms of its reproductive mechanism. Katagnymene retains trichomes that are segmented by separation discs within the mucilage (Table 2). Among the various species of Katagnymene, the vegetative cells of K. terrestris exhibit the lowest length-to-width ratio (Table 3).  The Katagnymene has been reported to be planktonic in the ocean or freshwater or inhabit attached to the substrates [4]; however, K. terrestris FBCC-A195 is collected in the soil of a stream floodplain. This habitat is the first report on the Katagnymene ecology. The width of mucilage of K. terrestris FBCC-A195 is thicker compared to freshwater species (K. accurata, K. mucigera, K. palustris, K. spirulinoides) ( Table 3).
This study proposed Katagnymene terrestris sp. nov. FBCC-A195 belonging to the family Gomontiellaceae through morphological features (using LM, and TEM), 16S rRNA, 16S-23S ITS sequences, and soil habitats in the Republic of Korea.