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

Abstract

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.

1. 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 data [3]. Additionally, the genus Katagnymene falls within a taxonomically ambiguous category phylogenetically [9].

Also, recently a novel classification system has been proposed, utilizing polyphasic analysis, to update the orders and families of cyanobacteria [9]. This classification is based on the genomic sequences of numerous cyanobacteria amassed to date, as well as the well-characterized reference strains. As a result, within this classification system, the order Oscillatoriales, which previously included the family Gomontiellaceae, has now been split into five orders, including the newly proposed order Gomontiellales [8,9].

In the Katagnymene, nine taxa have been reported worldwide, i.e., K. accurata by Geitler, K. bergii by Kiselev, K. maharashtrensis by Ashtekar and Kamat, K. mucigera by Compére, Komárek, Walmsley and Barlow, K. palustris by West, K. pelagica by Lemmermann, K. pelagica var. major by Wille, K. spiralis by Lemmermann, and K. spirulinoides by An [8,10]. The Katagnymene was reported by Lemmermann [10] based on two marine species descriptions, i.e., K. pelagica and K. spiralis. Then followed by freshwater species, K. palustris, K. maharashtrensis, K. mucigera, K. accurata, K. spirulinoides, and additional marine species, K. bergii. All species were identified and reported from the aquatic habitats. In terms of molecular phylogenetics, both K. pelagica and K. spiralis were the diazotroph with the nifH gene and showed a close relationship to the Trichodesmium by Ehrenberg ex Gomont [11]. The phylogeny using the hetR supported the monophyly of Trichodesmium including K. pelagica and K. spiralis [12,13]. However, there is no molecular phylogeny of Katagnymene and Trichodesmium using broad taxon sampling in Gomontiellaceae.

In the present study, we collected and cultured a strain of Korean soil cyanobacterium from the genus Katagnymene. They were compared morphologically using microscopy (light and transmission electron) and molecularly, and we propose a new species to science, Katagnymene terrestris sp. nov.

2. Materials and Methods

2.1. 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).

2.2. 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.

2.3. 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).

2.4. 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 (MC³) 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.

2.5. 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.

3. Results

3.1. 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.

3.2. 16S rRNA Characteristics and Phylogeny

The newly determined 16S rRNA of Katagnymene terrestris FBCC-A195 was 1474 bp with 54.8% GC content (A: 25.1%, C: 22.7%, G: 32.1%, T: 20.1%). The total alignment of 16S rRNA of 71 oscillatorian taxa and two outgroups was 1534 bp with 54.9% GC content (A: 25.2%, C: 23.1%, G 31.8%, T: 19.6%). The alignment contained 1008 constant sites (65.7%) and 526 variable sites (34.3%). The average p-distance of K. terrestris FBCC-A195 to gomontiellacean taxa was 0.061 (±0.039, standard deviation). Within the Gomontiellaceae, the p-distance ranged from 1.7% (25 nucleotide (nt) difference) between K. terrestris FBCC-A195 and Hormoscilla pringsheimii SAG 1407-1 to 12% (159 nt difference) between K. terrestris FBCC-A195 and Komvophoron kgarii Wanggoolba Creek (

Table 1. Genetic distance (p-distance) of Katagnymene terrestris FBCC-A195 from selected species of Gomontiellaceae and Microcoleaceae based on the partial 16S rRNA.

	Species (Acc. No.)	[1]	[2]	[3]	[4]	[5]	[6]	[7]	[8]	[9]	[10]	[11]	[12]	[13]	[14]	[15]
[1]	Katagnymene terrestris FBCC-A195 (MZ536612)
[2]	Crinalium epipsammum SAG 22.89 (NR_112218)	2.22
[3]	Crinalium magnum Hg-6-6 (MK211234)	2.19	0.97
[4]	Hormoscilla pringsheimii SAG 1407-1 (KM019982)	1.71	0.90	1.09
[5]	Starria zimbabweënsis SAG 74.90 (NR_112217)	3.18	3.46	3.39	3.25
[6]	Komvophoron hindakii clone K9_3P8 (KJ140092)	8.73	9.01	9.23	9.32	9.88
[7]	Komvophoron kgarii Wanggoolba Creek (NR_118555)	12.00	11.50	11.67	12.04	13.03	8.80
[8]	Trichodesmium contortum (AF013028)	10.25	10.24	9.83	10.24	10.80	12.58	13.63
[9]	Trichodesmium erythraeum SERB-14 (KM982563)	10.75	10.52	10.11	10.53	11.08	13.20	14.39	1.95
[10]	Trichodesmium hildebrandtii (AF091322)	10.62	10.54	10.27	10.54	11.17	12.61	13.97	2.09	2.38
[11]	Trichodesmium pelagicum str. JWI1 (AF518769)	13.54	13.41	13.76	13.74	13.54	17.83	16.56	1.52	2.06	0.51
[12]	Trichodesmium radians (AF013029)	10.38	10.23	9.82	10.24	10.65	12.73	13.91	0.84	1.95	2.23	2.24
[13]	Trichodesmium thiebautii (AF091321)	10.49	10.41	10.13	10.41	11.03	12.53	13.81	1.95	2.30	0.56	0.53	2.09
[14]	Dapis pleousa FFP12-2 (MF061797)	10.53	10.43	10.59	10.83	11.31	12.43	13.63	2.55	3.19	3.19	2.74	2.78	3.11
[15]	Dapis pnigousa NAB11-15 (MF061807)	10.12	9.78	9.95	10.19	10.74	12.13	13.62	2.55	3.10	3.10	2.88	3.10	3.03	1.83
		p-distance (%)

).

The ML phylogeny of 16S rRNA supports for close relationships of K. terrestris FBCC-A195 to Hormoscilla, Crinalium, and Starria (Clade CHKS in Figure 5; 1 BPP, 100% MLB, 100% NJB). Within the Gomontiellaceae, Komvophoron distinguished from other genera, the Clade CHKS with moderate supports (0.93 BPP, 65% MLB, 89% NJB). The present phylogeny supports close relationships of Desertifilaceae, Gomontiellaceae, and Wilmottiaceae (0.99 BPP, 83% MLB, 75% NJB), and a monophyly of Microcoleaceae (1 BPP, 95% MLB, 94% NJB).

Furthermore, through the AU test, we examined the relationship between K. terrestris FBCC-A195 and Trichodesmium pelagicum str. JWI1 by forcing them into a sister relationship. The hypotheses of placing K. terrestris FBCC-A195 within Gomontiellaceae (lnL = −11,774.28, p-value = 0) and Microcoleaceae (lnL = −11,877.80, p-value = 0) were both significantly rejected by the ML phylogeny. Additionally, the AU test indicated the possibility of six alternative topologies, including Desertifilaceae, Gomontiellaceae (Clade CHKS and Komvophoron), and Wilmottiaceae (Figure 6).

3.3. Secondary Structure of 16S–23S ITS Region

The complete 16S–23S ITS region of Katagnymene terrestris FBCC-A195 was sequenced. The predicted 16S–23S ITS secondary structure included the D1–D1’, Box-B, and V3 helices and was compared to relative cyanobacteria, including Crinalium and Hormoscilla (Figure 7).

The D1–D1’ helix of the K. terrestris FBCC-A195 showed a similar structure to other relative species. The total length of the D1–D1’ helix was 59-bp, and the basal stem consisted of a 4-bp helix (5′-GACC–GGUC-3′) in the K. terrestris FBCC-A195. A 7-bp 3′ side unilateral bulge was located as other relative species (Figure 7A–F). After the base position 15, however, K. terrestris FBCC-A195 showed different D1–D1’ helix structures to other strains. In addition, K. terrestris FBCC-A195 showed one mid-internal loop located at 18–22/36, and a 5-bp terminal loop (5′-UUAAU-3′; Figure 7A).

The total length of the Box-B helix was 42-bp, and the basal stem consisted of a 10-bp helix (5′-UAGCA–UGCUG-3′) in the K. terrestris FBCC-A195. The Box-B helix of K. terrestris FBCC-A195 was longer than other strains (33–34 bp; Figure 7G–L). The K. terrestris FBCC-A195 showed two mid-internal loops located at 6–7/36–37 and 13–16/28–30, and a 7-bp terminal loop (5′-ACAAUCA-3′; Figure 7G).

The V3 helix of the K. terrestris FBCC-A195 showed more difference in structure to other regions of relative strains (Figure 7M–R). The total base number of the V3 helix was 48-bp, and the basal stem consisted of a 10-bp helix (5′-GUCAA–UUGAC-3′) in the K. terrestris FBCC-A195. It was the shortest of any other strains. In addition, K. terrestris FBCC-A195 showed three mid-internal loops located at 6/42–43, 9–11/37–39, and 16–18/31–32, and a 4-bp terminal loop (5′-GAAA-3′; Figure 7M).

3.4. 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 Gomontiellales Strunecký and Mareš 2023.

Family Gomontiellaceae Elenkin ex Geitler 1942.

Genus Katagnymene Lemmermann 1899.

Katagnymene terrestris N. Lee, D. Kim, E.C. Yang, and O. Lee sp. nov. (Figure 2, Figure 3 and Figure 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. Morphological comparison in Gomontiellaceae and Trichodesmium (Microcoleaceae).

Genus	Cross Section	Fragmentation	Mucilage	Colony	Thylakoids
Crinalium	Flattened, Oval to cylindrical (with rounded ends)	Hormogonia	Envelope	Irregular agglomerations	Helically twisted Swirl-like
Gomontiella	C-shaped	Hormogonia	-	Solitary	Swirl-like or parietal
Hormoscilla	Cylindrical, circular	By necridia	Without	Irregular agglomerations	Swirl-like or parietal
Katagnymene	Cylindrical, circular	Mucilaginous separation disk or necridic cell	Envelope	Solitary	Irregularly dispersed Swirl-like structures
Starria	Triangular, triradiate	Without necridic cell	-	Irregular clusters	Swirl-like structures
Trichodesmium	Cylindrical, circular	Middle of trichomes	Envelope	Parallel or radially arranged	Radial thylakoid with gas vesicle

-: no data available.

). Among the various species of Katagnymene, the vegetative cells of K. terrestris exhibit the lowest length-to-width ratio (

Table 3. Morphological characteristics of Katagnymene.

Species	Filaments	Envelopes	Trichome	Cells	Apical Cells	Ecology
Katagnymene terrestris FBCC-A195	Straight or bent	50.0–53.0 μm diffluent, thick	W. 4.0–14.6 slightly constrictions at cross walls	L. 1.35–1.54	Rounded, truncate, convex, O	Soil
K. accurata	Screw-like	2.2–2.5 (–5.5) μm	W. 9.5–11.0 slightly constrictions at cross walls	L. 1.4–2.6	Convex, rounded, X	Freshwater
K. bergii	Straight	75.0–90.0 μm	W. 20.0 not constrictions	L. 2.5–3.0	Flattened, O	Ocean
K. mucigera	Straight or flexuous	- μm thick	W. 10.0–14.0 constrictions at cross walls	L. 2.5–7.0	-	Freshwater
K. palustris	Slightly curved	- μm thick	W. 28.0 not constrictions	L. 2.8–3.0	Truncate, X	Freshwater
K. pelagica T	Straight or bent	93.0–100.0 μm very wide	W. 16.0–27.0 not or slightly constrictions	L. 3.0–4.0	Rounded, O or X	Ocean
K. spiralis	Irregularly	150.0–168.0 μm	W. 20.0–22.0 not constrictions	L. 3.0–4.0	Rounded -	Ocean
K. spirulinoides	Regularly or loosely spirally	17.0–18.0	W. 2.5 -	L. -	Conical -	Freshwater

^T: type species, -: no data available, O: calyptra presence, X: calyptra absence.

).

Etymology: The specific epithet “terrestris” refers to the habitat preference of the studied cyanobacterium, which primarily inhabits soil substrates.

Holotype (designated here): A formaldehyde fixed specimen, NIBRCY0000001277 in the Herbarium at the National Institute of Biological Resources (KB), from reference strain FBCC-A195.

Reference strain: FBCC-A195.

Type locality: Soil between rocks underneath the Jangnak Bridge, situated along the Yeo Stream in the Republic of Korea (37°17′21.9″ N 127°03′41.1″ E).

Habitat: Inhabit the terrestrial environment within the soil located between rocks in temperate regions.

Gene sequences: 16S rRNA, 16S–23S ITS, and 23S rRNA gene sequences with GenBank Accession number of MZ536612.

4. Discussion

The Gomontiellaceae contains Crinalium, Gomontiella, Hormoscilla, Katagnymene, Komvophoron, and Starria identified with specific cross-section shapes of the cell and mucilage surrounding the trichome [4,8,27]. The trichome of K. terrestris FBCC-A195 shows a circular cross-section shape and surrounding mucilage; these morphologies are congruent with Katagnymene (Figure 3). K. terrestris FBCC-A195 is similar to K. pelagica, the type species of Katagnymene, and is clearly distinguished by (i) the shorter cell length than width and (ii) the narrower mucilage layer (Table 3). The thylakoids show a swirl-like pattern which is a diagnostic characteristic of the Gomontiellaceae [3]. The cell possesses pit-like pores and junctional pores in the cell wall (Figure 4). Mucous microfibrils in the cross-cell wall of the Crinalium magnum by Fritsch and John were also identified in the K. terrestris FBCC-A195 (Figure 3) [2]. The K. terrestris FBCC-A195 was clearly identified by its morphological characteristics and deposited in public culture collection for the first time. A few strains are available in algal culture collections in the world, such as Hormoscilla pringsheimii SAG 1407-1, H. pringsheimii CCALA 1054, Crinalium epipsammum SAG 22.89, C. magnum SAG 34.87, and Starria zimbabweënsis SAG 74.90.

Taxonomic research on novel cyanobacteria has been growing rapidly since the introduction of molecular methods [3]. A polyphasic approach is considered useful for classifying cyanobacteria families including 16S rRNA phylogeny [16,18,28]. In the present study, the 16S rRNA phylogeny of the Gomontiellaceae was congruent with previous studies [1,2]. Monophyly of the Crinalium, Hormoscilla, Katagnymene, and Starria (Clade CHKS in Figure 5) supported with maximum values in all analyses (BI, ML, and NJ searches), although genus-level relationships were not fully resolved. Additional sequence data are needed to resolve the generic relationships within the Gomontiellaceae such as a type species of Gomontiella.

Lundgren et al. [13] identified marine planktonic cyanobacteria K. pelagica and K. spiralis and argued that both species K. pelagica/spiralis JWI1 should be transferred to Trichodesmium based on the phylogenies using 16S rRNA and hetR. However, among strains identified as K. pelagica/K. spiralis strains (PLA1, F47-6, A32-1, F8-8, B51-2, B51-2, B41-1, A27, F8-6, F8-1, F8-3, F8-5, B41-2, JWI1, A25), K. spiralis JWI1 was the only taxa with the 16S rRNA data (584 bp). In the present study, we tested the taxonomic position of Katagnymene by using AU tests. The genus was considered a monophyletic using the enforced sister relationship of K. terrestris FBCC-A195 and K. spiralis str. JWI1 (AF518769), then positioned in families Gomontiellaceae or Microcoleaceae. Both hypotheses, i.e., monophyletic Katagnymene as a member of Gomontiellaceae (lnL = −11,774.28, p-value = 0) and that of Microcoleaceae (lnL = −11,877.80, p-value = 0), were rejected significantly to the ML phylogeny. These results support the best phylogeny that K. terrestris FBCC-A195 and T. pelagicum str. JWI1 are not a sister taxon.

In the ML phylogeny resolved a few family level relationships, i.e., the monophyly of Desertifilaceae, Gomontiellaceae (Clade CHKS and Komvophoron), and Wilmottiaceae. However, the AU test showed that six possible alternative topologies were likely (Figure 6). For example, the sister relationship of Komvophoron and Desertifilaceae (T01: p-value = 0.062, T04: p-value = 0.46, and T11: p-value = 0.07), the alternative relationships of Desertifilaceae and Wilmottiaceae (T09: p-value = 0.19 and T12: p-value = 0.156), and non-monophyly of Clade CHKS and Komvophoron (T3: p-value = 0.327). These results provide evidence for the distinction of Komvophoron, a possible novel branch [7].

The ITS secondary structure (D1–D1ʹ, Box-B, and V3 helix) of K. terrestris FBCC-A195 was compared with genera belonging to the Gomontiellaceae [2]. A few conserved nucleotides of K. terrestris FBCC-A195 are different from known genera in Gomontiellaceae, i.e., Crinalium and Hormoscilla. Additional mid-internal loops in the Box-B helix show a unique genetic characteristic of K. terrestris FBCC-A195 (Figure 7).

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.