# Expanding species diversity in the monotypic genera Thalassoporum and Tumidithrix (Pseudanabaenales, Cyanobacteriota) with the description of Thalassoporum mexicanum sp. nov. and Tumidithrix helvetica sp. nov

Aniket Saraf, Boris Aleksovski, Eddy Blondet, Svetislav Krstić, Alexis

Criscuolo, Muriel Gugger

► To cite this version:

Aniket Saraf, Boris Aleksovski, Eddy Blondet, Svetislav Krstić, Alexis Criscuolo, et al. Expanding species diversity in the monotypic genera Thalassoporum and Tumidithrix (Pseudanabaenales, Cyanobacteriota) with the description of Thalassoporum mexicanum sp. nov. and Tumidithrix helvetica sp. nov. International Journal of Systematic and Evolutionary Microbiology, 2025, 75 (8), 10.1099/ijsem.0.006869. pasteur-05233284

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'© [Saraf et al., 2025]. The definitive peer reviewed, edited version of this article is published

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in [International Journal of Systematic and Evolutionary Microbiology, 75, 8, 2025,

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https://doi.org/10.1099/ijsem.0.006869].'

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Expanding species diversity in the monotypic genera *Thalassoporum* and *Tumidithrix* (*Pseudanabaenales*, *Cyanobacteriota*) with the description of *Thalassoporum mexicanum* sp. nov. and *Tumidithrix helvetica* sp. nov.

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Aniket Sarafª, Boris Aleksovskiᵇ, Eddy Blondetª, Svetislav Krstićᵇ, Alexis Criscuoloᶜ, Muriel Guggerª*

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ª Institut Pasteur, Université Paris Cité, Collection of Cyanobacteria, F-75015 Paris, France

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ᵇ Institute of Biology, Faculty of Natural Sciences and Mathematics-Skopje, Ss. Cyril and

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Methodius University in Skopje, Arhimedova 3, PO Box 162, 1000 Skopje, North Macedonia

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ᶜ Institut Pasteur, Université Paris Cité, GIPhy - Genome Informatics and Phylogenetics,

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Biological Resource Center of Institut Pasteur, Paris F-75015, France

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* Corresponding author: Muriel Gugger

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E-mail : muriel.gugger@pasteur.fr

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ORCID ID: 0000-0001-6728-1976

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**Running title:** Novel species of *Thalassoporum* and *Tumidithrix*

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**Keywords:** *Thalassoporum*, *Tumidithrix*, *Pseudanabaenales*, basal cyanobacteria, *nif* gene

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cluster

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27 **Accession numbers:** AF091108, AM709633, AB039019, NC_019701, NC_019690,  
28 CBDDRA000000000

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30 **Author notes:** Two supplementary figures and two supplementary tables are available with the  
31 online version of this article.

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33 **Abbreviations:** AAI, average amino acid identity; ANI, average nucleotide identity; BIC, 34 Bayesian information criterion; BGC, biosynthetic gene cluster; COG, cluster of orthologous 35 groups; dDDH, digital DNA DNA hybridization; ITS, internal transcribed spacer; ME, 36 minimum evolution; ML, maximum likelihood; MP, maximum parsimony; NFGC, *nif* gene 37 cluster; NRPS, non-ribosomal peptide synthetase; OGRI, overall genome relatedness indices; 38 PKS, polyketide synthase; RiPPS, ribosomally synthesized and post-translationally modified 39 peptides. 40

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41 **Data availability:** The raw sequencing reads of strains PCC 7367T and PCC 7403T are 42 available under the run accession numbers SRR3926589-SRR3926590 (BioProject 43 PRJNA158713) and ERR14790590 (BioProject PRJEB87789), respectively. The 16S rRNA 44 gene sequences are available under the GenBank/EMBL/DDBJ accession numbers AF091108, 45 AM709633 (PCC 7367T) and AB039019 (PCC 7403T). The RefSeq accession numbers for the 46 complete genome sequences of PCC 7367T are NC_019701 and NC_019690 (assembly 47 GCF_000317065). The draft genome sequences of PCC 7403T are available under the 48 GenBank/EMBL/DDBJ accession number CBDDRA000000000 (assembly 49 GCA_965232925).
50 Abstract

51 The taxonomic identity of two well-documented pseudanabaenalean strains, *Pseudanabaena* 52 sp. PCC 7367T and *Pseudanabaena* sp. PCC 7403T, isolated from the samples collected from 53 the surface of a snail shell in an intertidal zone and sphagnum bog, respectively, were 54 reassessed using a polyphasic approach. The results from the morphological, phylogenetic, ITS 55 rRNA and genomic analyses confirmed PCC 7367T (= BACA0810T) and PCC 7403T (= 56 BACA0811T) represent novel species of the monotypic pseudanabaenalean genera 57 *Thalassoporum* and *Tumidithrix*, respectively, for which we propose the names *Thalassoporum* 58 *mexicanum* sp. nov. and *Tumidithrix helvetica* sp. nov. in accordance with the International 59 Code of Nomenclature of Prokaryotes. The analysis of the 16S-23S ITS rRNA region, a widely 60 used genetic marker for the delineation of closely related species, effectively distinguished the 61 *Thalassoporum* species. However, the closely related species within *Tumidithrix* could not be 62 demarcated using the currently accepted nucleotide dissimilarity cutoff for species delineation 63 but were clearly distinguished through genomic analyses. Accordingly, a revised ITS rRNA 64 nucleotide dissimilarity cutoff is proposed for species delineation within the genus *Tumidithrix*. 65 Furthermore, genomic investigations revealed that the predicted biosynthetic gene clusters in 66 the genome assemblies of PCC 7367T and PCC 7403T were not associated to any known natural 67 products. 68
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## 1. Introduction

The implementation of phylogenetic analysis has revolutionized cyanobacterial taxonomy resulting in the description of novel genera and species at a rapid pace (1-3). Many traditional genera, such as *Synechococcus* Nägeli, *Lyngbya* Agardh ex Gomont, *Nostoc* Vaucher ex Bornet and Flahault, *Calothrix* Agardh ex Bornet and Flahault, have been shown to be polyphyletic, leading to the description of numerous cryptic genera (4-9). One such genus is *Pseudanabaena*, established by Lauterborn (1915) to accommodate homocytic filamentous cyanobacteria with creeping movements and prominently constricted cells (10). At the time of description, two morphologically different species were included in the genus *Pseudanabaena*: *Pseudanabaena constricta* (Szafer) Lauterborn (Basionym: *Oscillatoria constricta*) and *Pseudanabaena catenata* Lauterborn (10). The taxonomic confusions over *Pseudanabaena* began immediately after its establishment, mainly due to the inclusion of *P. constricta*. In 1925, Geitler transferred *P. constricta* to the genus *Anabaena*, but later questioned the validity of the genus *Pseudanabaena*, and placed it in "anhang" at the end of Oscillatoriaceae (11, 12). In 1959, Desikachary recognized *Pseudanabaena* to be a valid genus and proposed *P. catenata* as the lectotype for the genus (13). This settled the debate around the taxonomic status of *Pseudanabaena*, and thereafter the genus was recognized as taxonomically valid in other classification schemes (1, 2, 14, 15).

Lauterborn's (10) definition of *Pseudanabaena* lacked any prominent diacritical features, which led the genus definition being revised multiple times (13, 14, 16, 17). Nevertheless, the genus remains poorly defined with lack of prominent diacritical features, grouping thin (< 3 µm) filamentous cyanobacteria having creeping movements and prominently constricted adjacent cells, that are connected with so called "hyaline bridges", a morphotype highly ubiquitous in the environment worldwide (11, 13, 14, 18). Consequently, several phylogenetic analyses demonstrated the genus to be polyphyletic (19-25). Recent advances in the taxonomic
studies on *Pseudanabaena*-like cyanobacteria has led to the description of four novel genera, namely *Pinocchia* Dvořák, Jahodářová & Hassler (20), *Stenomitos* Miscoe & Johansen (23), *Thalassoporum* Konstantinou & Gkelis (21) and *Tumidithrix* Luz, Kaštovský, Johans & Gonçalves (22). According to the most recent classification scheme, *Thalassoporum* and *Tumidithrix* belong to the order *Pseudanabaenales*, whereas *Pinocchia* and *Stenomitos* are placed in *Leptolyngbyales* (1).

*Thalassoporum* (type species – *Thalassoporum komarekii* corrig. Konstantinou and Gkelis) was established by Konstantinou et al. (21) to accommodate *Pseudanabaena*-like cyanobacteria that were epizoic or symbiotic, ecologically restricted to marine and brackish habitats, and phylogenetically distinct from *Pseudanabaena sensu stricto*. Similarly, *Tumidithrix* (type species – *Tumidithrix elongata* Luz, Kaštovský, Johans & Gonçalves) was also established as a phylogenetically distinct genus having *Pseudanabaena* and *Arthronema* Komárek & Lukavský like characters (22). Notably, the presence of swollen or involuted cells in *Tumidithrix elongata* was considered as one of the characteristic features. However, Aleksovski et al. (26) also reported swollen and elongated cells in *Pseudanabaena pruinosa* Aleksovski, Krstić, Komárek & Strunecký, making this character non-exclusive to *Tumidithrix*. Currently, *Thalassoporum* and *Tumidithrix* are monotypic. However, they contain multiple strains that have not been identified at the species level and require reevaluation to better understand the true species diversity in these basal cyanobacterial lineages (21, 22, 27). Among these strains are *Pseudanabaena* sp. PCC 7367T and *Pseudanabaena* sp. PCC 7403T that belong to the genera *Thalassoporum* and *Tumidithrix*, respectively (21, 22, 27). Previous morphological and ultrastructural analyses on these strains have significantly contributed to our understanding of the phenotypic characteristics of *Pseudanabaena* and *Pseudanabaena*-like taxa (16). However, the species-level identity of PCC 7367T and PCC 7403T remains unknown, highlighting the need for a detailed taxonomic study. Therefore, in this study we
reassessed *Pseudanabaena* sp. PCC 7367T and *Pseudanabaena* sp. PCC 7403T using a polyphasic approach. Our results confirm both strains to be novel species of these two monotypic genera for which the names *Thalassoporum mexicanum* sp. nov. and *Tumidithrix helvetica* sp. nov. are proposed under the provisions of the International Code of Nomenclature of Prokaryotes.

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## 2. Materials and methods

### 2.1 History of strains

Strain PCC 7367T was isolated by J. B. Waterbury in 1971 from a sample collected from the surface of a snail shell in an intertidal zone in Puerto Penasco, Mexico, whereas strain PCC 7403T was isolated by R. Rippka in 1972 from a sample collected from a sphagnum bog near Kastanienbaum, Vierwaldstättersee, Switzerland. Monoclonal cultures of PCC 7367T and PCC 7403T were obtained in 1973 and 1974, respectively (for detailed protocol, see Haande et al. (28). Since then, both strains have been maintained axenically at the Pasteur Cultures of Cyanobacteria (PCC) collection, Paris, France.

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### 2.2 Culture conditions

Both strains are maintained in a culture room at 22 °C illuminated with 5 µmol photons m⁻² s⁻¹ of daylight (cool white OSRAM L 18/640) with a photoperiod of 13/11 h of light/dark cycle.

Strain PCC 7367T is subcultured monthly in ASN-III medium, while PCC 7403T is subcultured bimonthly in BG11 medium (17).

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### 2.3 Morphological examination

Morphological evaluation of the strains was conducted using a Zeiss Axioskop2 microscope (Zeiss, Oberkochen, Germany) fitted with a Leica MC170 HD camera (Leica, Wetzlar,

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Germany) for microphotographs. Morphological characteristics such as the length of the trichomes, size and shape of cells, constrictions between the cells and presence of swollen or elongated cells were carefully examined. The samples for microscopic observation were prepared using 10% glycerol as the mounting medium. Approximately 100 cells were examined to determine their dimensions.

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## 2.4 Genomic DNA extraction, whole genome sequencing and assembly

The methodology for DNA extraction, whole genome sequencing, assembly and annotation for PCC 7367T was previously published (29). Similar DNA extraction protocol was used for strain PCC 7403T. The whole-genome sequencing of PCC 7403T was carried out with a Genome Sequencer Illumina HiSeq using the Standard Genomic library at Eurofins Genomics Europe Sequencing GmbH (Germany) and a 2×150 bp paired-end protocol, yielding 6,241,823 read pairs (331× sequencing depth and 396 bp insert size, on average). Read processing and genome assembly were performed using fq2dna v24.02 (gitlab.pasteur.fr/GIPhy/fq2dna). Completeness and contamination indices of the whole genome assemblies were estimated using CheckM v1.1.3 (30).

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## 2.5 Genomic investigation

Gene prediction and annotation of the PCC 7403T assembled scaffolds were performed using bakta v1.9.3 (31). Predicted coding sequences were classified into functional clusters of orthologous groups (COG) categories using COGniz v1.1 (gitlab.pasteur.fr/GIPhy/COGniz) with the 2020 update of the COG database (32). Biosynthetic gene clusters for secondary metabolites were inferred using antiSMASH v7.1.0.1 (33). Average nucleotide and amino acid identity (ANI and AAI, respectively) values were estimated using OGRI_B v1.2 (gitlab.pasteur.fr/GIPhy/OGRI).

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173 2.6 Phylogenetic analyses

174 The Sanger-sequenced 16S rRNA gene segments of PCC 7367T (accessions AF091108 and
175 AM709633) and PCC 7403T (AB039019) were gathered from the GenBank repository,
176 together with segments from their respective genome assemblies (locus tags:
177 PSE7367_RS09835 for PCC 7367T and TUMEXPCC7403_25600 for PCC 7403T). These
178 sequences were first aligned using MAFFT v7 (34) together with 63 other 16S rRNA segments
179 representative of the order Pseudanabaenales and other basal cyanobacterial genera (including
180 three sequences of Gloeobacter as an outgroup). The multiple sequence alignment was
181 manually curated to ensure that the folded secondary structure of the 16S rRNA gene was
182 maintained, leading to a total of 1,471 aligned nucleotide characters. A maximum likelihood
183 (ML) phylogenetic tree was inferred using IQ-TREE v2.4.0 (35) with evolutionary model
184 TIM3+F+R3, derived by minimizing the Bayesian information criterion (BIC). Branch
185 supports were assessed based on 500 bootstrap replicates and three different phylogenetic
186 reconstruction criteria: maximum parsimony (MP), minimum evolution (ME) and maximum
187 likelihood (ML) using TNT v1.5 (36), FastME v2.1.6.4 (37) and IQ-TREE v2.4.0,
188 respectively.

189 A multilocus phylogenetic tree was also inferred from 25 genomes using the 120 loci suggested
190 by Parks et al. (38). For each locus, amino acid sequences were gathered using tBLASTn and
191 aligned using MAFFT v7.505 (39, 40). Aligned characters were processed using BMGE v2.0
192 (41) to select those suited for phylogenetic analysis. ML phylogenetic inference from the
193 concatenation of the resulting 120 multiple sequence alignments (44,158 aligned characters)
194 was carried out using IQ-TREE v2.4.0 with evolutionary model LG+F+I+R4, derived by
195 minimizing the BIC. Bootstrap-based branch supports were assessed in the same way as the
196 16S rRNA phylogenetic tree.
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2.7 16S-23S ITS rRNA region analysis

199 The 16S-23S ITS rRNA regions were recovered from the assembled genomes of PCC 7367T
200 and PCC 7403T and compared with the ITS rRNA sequences of *Thalassoporum komarekii*
201 TAU-MAC 1515T and *Tumidithrix elongata* BACA0141T, respectively. The length of the D1-
202 D1', V2, BoxB and V3 helices, the presence or absence of tRNA<sup>IIe</sup> and tRNA<sup>Ala</sup>, and the
203 position of the spacer regions were assessed to determine the type of ITS rRNA region (42, 43)
204 . The folded secondary structures corresponding to the D1-D1', V2, BoxB and V3 helices were
205 determined using Mfold (44) hosted on the UNAFold web server (www.unafold.org). The
206 Pairwise nucleotide dissimilarities (*p*-distances) were computed using MEGA X (45).

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3. Results and discussion

209 The order *Pseudanabaenales*, as delineated by (1), forms a monophyletic lineage. However,
210 significant taxonomic confusion exists within the order, primarily due to the polyphyly of the
211 genus *Pseudanabaena* (20, 23-25). Recent 16S rRNA gene-based phylogenetic analyses
212 demonstrated that the two strains PCC 7367T and PCC 7403T (previously identified as
213 *Pseudanabaena* based on morphological characteristics) belong to the newly described genera
214 *Thalassoporum* and *Tumidithrix*, respectively (21, 22). However, as the species level identity
215 of both strains remains unclear, this study aimed to determine the species status of PCC 7367T
216 and PCC 7403T using a combination of morphological, phylogenetic, genomic and ITS rRNA
217 analyses.

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3.1 Polyphasic evaluation of PCC 7367<sup>T</sup>

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3.1.1 Morphological analysis
PCC 7367<sup>T</sup> exhibited short (3–10 cells), uniseriate, isopolar and straight trichomes. In the liquid medium, the strain grows by attaching to the bottom of the flask and has creeping growth on solidified medium. This strain is currently non-motile, and this observation is in agreement with the study of Guglielmi and Cohen-Bazire (16). The trichomes of PCC 7367<sup>T</sup> were deeply constricted with thin but evident “hyaline bridges” (Fig. 1a). The constrictions reduce the cell diameter by 70–80%, resulting in a transverse septum to only 20% of the trichome diameter (16). These transverse partitions were as circular disks and almost entirely situated around the central perforation (16). The cells were usually barrel-shaped or cylindrical with rounded ends, (1.4)1.9–3.7(4.1) µm long and (1.1)1.3–1.9(2.1) µm wide (Fig. 1b). The cross-walls were 3–6 times thicker than the parietal peptidoglycan with single perforation (16). The ultrastructure analysis of PCC 7367<sup>T</sup> confirms the presence of polar gas vesicles (Fig. 9 and 10 from (16); online resource 3 from (46)). Some trichomes exhibited highly constricted spherical cells that were shorter than wide. Occasionally, these spherical cells were concatenated resulting in a moniliform region in the trichome (Fig. 1d). The ends of the apical cells were flat-rounded or conical-rounded (Fig. 1a).

The trichomes were without a prominent sheath. However, in the ultrastructure analyses, PCC 7367<sup>T</sup> was sometimes shown to develop a discontinuous envelope (Fig. 10 from (16)). The thylakoids were peripherally arranged and were 6–10 in number (Fig. 9 and 10 from (16)). The thylakoid membranes in PCC 7367<sup>T</sup> were prolonged and completely continuous around the cells as concentric circles, and were not interrupted in the region of the cell poles, leaving only a tiny area around the deep murein layer and the central perforation that can bear only a small number of gas vesicles (Fig. 9 and 10 from (16)). Reproduction is by intercellular trichome fragmentation and production of hormogonia (Fig. 1c). PCC 7367<sup>T</sup> synthesizes C-phycoerythrin but does not undergo complementary chromatic adaptation (16, 47, 48).
Based on the morphological characteristics, PCC 7367T was initially included in the genus *Pseudanabaena* (17). However, due to the presence of intensely deep constrictions and thylakoids at the cell's poles, PCC 7367T was further grouped in a different phenon from the one comprising PCC 7408 (reference strain of *P. catenata* - type species of *Pseudanabaena*) and PCC 7429 (reference strain for *P. biceps*) (16). Later, PCC 7367T was confirmed to be different from true *Pseudanabaena* species and grouped the strain in the *Pseudanabaena* cluster 2, distinct from typical *Pseudanabaena* (18). Recently, PCC 7367T was shown to be a member of the monotypic genus *Thalassoporum* (21). Morphologically, it satisfies the generic description of *Thalassoporum* and shows similarities with the type species – *Thalassoporum komarekii* TAU-MAC 1515T. However, the higher number of thylakoids and the presence of moniliform region differentiates PCC 7367T from *Thalassoporum komarekii* TAU-MAC 1515T (Table 1).

### 3.1.2 Genome features

The genome assembly of strain PCC 7367T (29) consists of a 4,557,046 bp chromosome (NC_019701) and a 328,634 bp plasmid (NC_019690), with a G+C content of 46.22%. Completeness and contamination indices of the genome assembly were 98.23% and 0.59%, respectively, indicating a high-quality genome according to the recommendations of Riesco and Trujillo (49). Gene prediction led to the inference of 6 rRNAs, 46 tRNAs and 3,881 coding sequences. Among the predicted COGs, a high abundance of genes was assigned to signal transduction mechanisms [T], followed by energy production and conversion [C], cell wall/membrane/envelope biogenesis [M] and translational, ribosomal structure and biogenesis [J] (Supplementary Fig. S1). The Biosynthetic Gene Cluster (BGC) prediction for the synthesis of natural products resulted in the identification of two regions with ribosomally synthesized and post-translationally modified peptides (RiPPs) clusters. However, these
clusters had no resemblance to known clusters in the current antiSMASH database, potentially coding for terpene and resorcinol. As the clade of *Pseudanabaena* is poorly dotted in BGCs (29), the only *Thalassoporum* genome at the root of the *Pseudanabaena* clade also encodes few BGCs.

### 3.1.3 Phylogenetic analyses

In the multilocus phylogenetic tree, PCC 7367<sup>T</sup> clustered independently at the base of the order *Pseudanabaenales*, distinct from *Pseudanabaena sensu stricto* and *Tumidithrix* (**Fig. 2**). A similar clustering pattern was reported by Strunecky et al. (1). Based on their multilocus and 16S rRNA gene phylogenetic classifications, the family Thalassoporaceae, was established consisting of a single monotypic genus *Thalassoporum*, to accommodate marine pseudanabaenalean strains (1). Our multilocus phylogenetic analysis, with higher taxa within *Pseudanabaenales*, supports the creation of Thalassoporaceae. However, in our multilocus phylogenetic tree, the genus *Thalassoporum* is currently represented by only PCC 7367<sup>T</sup> due to unavailability of the genome of the type species *Thalassoporum komarekii* TAU-MAC 1515<sup>T</sup>.

In the 16S rRNA gene phylogenetic tree, PCC 7367<sup>T</sup> clustered within the genus *Thalassoporum* with strong support at a node consisting of *Thalassoporum komarekii* TAU-MAC 1515<sup>T</sup> (**Fig. 3**). The genus *Thalassoporum* formed a strong and distinct clade within the order *Pseudanabaenales*, with all *Thalassoporum* strains being isolated from marine habitat, in coherence with previous studies (1, 21, 50). Furthermore, the nucleotide similarity of the 16S rRNA gene within the genus *Thalassoporum* ranged from 95.2%–99.1%, and the similarity between PCC 7367<sup>T</sup> and its phylogenetically closest strain (*Thalassoporum komarekii* TAU-MAC 1515<sup>T</sup>) was 99.1%, higher than the suggested species delineation threshold of 98.7% in prokaryotes (51).
298 Since the analysis of the 16S rRNA gene is often inconclusive in delineating closely related
299 species within various cyanobacterial genera, we also analyzed the 16S-23S ITS rRNA, as it is
300 reported to be an effective tool for delineating closely related species if used diligently (42, 43,
301 52-55). Two identical ITS rRNA operons (accession number: NC_019701; operon 1: 2486223-
302 2486733; operon 2: 1761656-1762166) having both tRNA genes (tRNA<sup>Ile</sup> and tRNA<sup>Ala</sup>) were
303 recovered from the genome assembly of PCC 7367<sup>T</sup>. The comparative sequence analysis of the
304 D1-D1', V2, BoxB and V3 helices, the presence of tRNA<sup>Ile</sup> and tRNA<sup>Ala</sup>, and the position of
305 the spacer regions indicated that the ITS rRNA sequences of PCC 7367<sup>T</sup> and *Thalassoporum
306 komarekii* TAU-MAC 1515<sup>T</sup> were of the same type (**Supplementary Table S1**), an essential
307 criterion to conduct ITS rRNA analyses (42, 43).

308 The folded secondary structure of D1-D1' helix of PCC 7367T consists of a five bp basal clamp
309 followed by a six nucleotides basal unilateral bulge opposed by a single nucleotide, an internal
310 helix with two unilateral bulges and a terminal bilateral bulge with 14 nucleotides (Fig. 4). The
311 difference in the number of nucleotides in the basal unilateral bulge and the terminal bilateral
312 bulge differentiated the secondary structure of PCC 7367T from TAU-MAC 1515T. The V2
313 helix in PCC 7367T and TAU-MAC 1515T contained only four nucleotides which were
314 identical sequences (Supplementary Table S1). The folded secondary structure of the BoxB
315 helix of PCC 7367T consisted of a five bp basal clamp followed by two bilateral bulges, an
316 internal helix with a single unilateral bulge and terminal bilateral bulge (Fig. 4). This structure
317 was completely different from the folded secondary structure of the BoxB helix of TAU-MAC
318 1515T.

319 The folded secondary structure of V3 helix of PCC 7367T consisted of two bp basal clamp
320 followed by two bilateral bulges (Fig. 4). This structure was similar to the secondary structure
321 of the V3 helix of TAU-MAC 1515T, with a minor difference in the topology resulting from
322 the substitution of guanine by uracil. The nucleotide dissimilarity between the ITS rRNA
323 sequence of PCC 7367T and TAU-MAC 1515T was 12.9%, higher than the commonly used
324 threshold of >7% for cyanobacterial species demarcation (53). The comparative secondary
325 structure analysis of the D1-D1' and BoxB helices of the ITS rRNA and the observed nucleotide
326 dissimilarity of 12.9% confirmed PCC 7367T to be a novel species of the genus *Thalassoporum*
327 for which we propose the name *Thalassoporum mexicanum* sp. nov.

328

329 **3.2 Polyphasic evaluation of PCC 7403<sup>T</sup>**

330

331 **3.2.1 Morphological analysis**

332 Strain PCC 7403T exhibited long, uniseriate, isopolar, straight or irregularly curved trichomes,
333 always without sheaths (Fig. 5a-b). In liquid medium, the strain grows in the form of dispersed
334 colonies and has a mat-like appearance on the solidified medium. The strain is currently non-
335 motile, and this observation is in coherence with Guglielmi and Cohen-Bazire (16). The
336 trichomes were intensely constricted with thin but evident "hyaline bridges" (Fig. 5a-b). The
337 constrictions and transverse partitions in PCC 7403T were similar to PCC 7367T (16). Previous
338 studies reported short (3-6 cells) trichomes in PCC 7403T (16-18); however, we observed
339 longer trichomes in our culture condition (Fig. 5a-f). The cells were usually cylindrical and
340 occasionally elongated (Fig. 5a-b). The elongated cells were sometimes swollen (Fig. 5a-b).
341 The cross-walls were 3-6 times thicker than the parietal peptidoglycan with single perforation
342 (16). The cylindrical cells were 2.3-6.7 µm long and 1.3-2.4 µm wide, whereas the elongated
343 cells were 7.0-17.5 µm long and 1.7-2.3 µm wide. Notably, some cells displayed aberrant cell
344 morphologies (Fig. 5d-e). Some trichomes exhibited barrel-shaped cells that were
345 concatenated resulting, in the moniliform region (Fig. 5c).
The ultrastructure analysis of PCC 7403<sup>T</sup> confirmed the presence of gas vesicles on either side of transversal septa, polyphosphate granules and 4-8 peripherally arranged thylakoids (Fig 4 in Guglielmi & Cohen-Bazire (16)). Unlike PCC 7367<sup>T</sup>, the thylakoid membrane in PCC 7403<sup>T</sup> was not continuous at the cell's poles. Reproduction in PCC 7403<sup>T</sup> was by intercellular trichome fragmentation and formation of hormogonia. The hormogonium cells were smaller than the cells of the mature filaments (Fig. 5f-i). PCC 7403<sup>T</sup> neither synthesizes C-phycoerythrin nor undergo complementary chromatic adaptation (48). Furthermore, PCC 7403<sup>T</sup> synthesizes two phycocyanin subunits (α and β) as opposed to the four phycocyanin subunits (α1, α2, β1 and β2) synthesized by *Pseudanabaena* sensu stricto strains (48).

Several studies included PCC 7403<sup>T</sup> as a representative of the genus *Pseudanabaena* (16, 17). However, based on the differences in the phycobilisome composition and low DNA-DNA hybridization percentages, PCC 7403<sup>T</sup> was further excluded from the revised definition of *Pseudanabaena* sensu Guglielmi and Cohen-Bazire (48). Later, PCC 7403<sup>T</sup> was confirmed to be distinct from true *Pseudanabaena* and the strain was categorized as a representative of *Pseudanabaena* cluster 4 (18). Recently, PCC 7403<sup>T</sup> was shown to be member of the monotypic genus *Tumidithrix* and the strain showed morphological similarity with the type species – *Tumidithrix elongata* BACA0141<sup>T</sup> with respect to the trichome length, absence of sheath, elongated swollen cells and aberrant cell morphology. However, the presence of moniliform region and shorter length of the elongated cells (17.5 µm vs. 30 µm) differentiates PCC 7403<sup>T</sup> from *Tumidithrix elongata* BACA0141<sup>T</sup> (Table 2).

### 3.2.2 Genome features

The genome assembly of PCC 7403<sup>T</sup> is 5,693,047 bp in length on 81 contigs, with a G+C content of 45.30%. Completeness and contamination indices of the genome assembly were 98.58% and 0.47%, respectively, indicating a high-quality genome (49). Gene prediction led
371 to the inference of 3 rRNAs, 74 tRNAs and 5,059 coding sequences. Among the predicted
372 COGs, a high abundance of genes was assigned to signal transduction mechanisms [T],
373 followed by energy production and conversion [C], cell wall/membrane/envelope biogenesis
374 [M] and coenzyme transport and metabolism [H] **(Supplementary Fig. S2)**. The BGC
375 prediction for the synthesis of natural products resulted in the identification of five regions.
376 One polyketide synthase (PKS) cluster was similar at 48.4% to the CF1 in *Gloeobacter*
377 *violaceus* PCC 7421(56), coding for unknown glycolipid. As this modified 'heterocyte
378 glycolipid' cluster is now found three times outside the heterocyte-producing lineage
379 (*Gloeobacter violaceus* PCC 7421<sup>T</sup>, *Gloeothece* sp. PCC 7428 and *Tumidithrix helvetica* PCC
380 7403<sup>T</sup>), it might be possible to investigate the diversity of polyunsaturated fatty acids produced.
381 A non-ribosomal peptide synthetase (NRPS) module was also present but without resemblance
382 to a previously described BGC. Also, three RiPP-like clusters were present, but they were too
383 partial (1 to 3 genes-long). Thus, none of these BGCs were associated with any of the known
384 natural products.

385 PCC 7403<sup>T</sup> was shown to perform N<sub>2</sub> fixation under anaerobiosis (57) but the genetic
386 organization of the *nif* gene cluster (NFGC) in this strain is unknown. *nifHDK* operon which
387 encodes for the structural components (dinitrogenase reductase and dinitrogenase) was found
388 in its genome assembly, together with *nifENXW*, *nifBSU* and *nifVZT* operons (**Fig. 6**). Notably,
389 NFGC was not found in the genome assembly of *Tumidithrix elongata* BACA0141<sup>T</sup>. However,
390 a few other pseudanabaenalean strains (PCC 6802, SR411 and UBA1465) have been reported
391 to contain NFGC, indicating its sporadic distribution in the order *Pseudanabaenales*.

392 The genetic organization of the *nif* gene cluster in PCC 7403<sup>T</sup> belongs to type F, as defined by
393 Chen et al. (58). Among the other pseudanabaenalean strains with NFGC, the genetic
394 organization in SR411 and UBA1465 was also of type F, whereas of type A in PCC 6802 (58).
395 Apart from pseudanabaenalean strains, NFGC type F is also reported from the strains of another
basal cyanobacterial order, *Thermostichales* (*Synechococcales* I (58)). However, the distribution of NFGC type F is not limited to basal cyanobacterial lineages, as it has also been reported from deeper lineages such as the orders *Synechococcales* and *Leptolyngbyales* (*Synechococcales* IV and V (58)). The sporadic distribution of NFGC, especially among the non-heterocytous cyanobacteria, and multiple genomic rearrangement events as evidenced by different structural types of NFGC within a single order (i.e. type A and F in *Pseudanabaenales*; **Fig. 6**) indicate a complex evolutionary trajectory of NFGC in Cyanobacteriota.

### 3.2.3 Phylogenetic analyses

In the multilocus phylogenetic tree, PCC 7403<sup>T</sup> and *Tumidithrix elongata* BACA0141<sup>T</sup> formed a distinct and strongly supported clade within the family Pseudanabaenaceae (**Fig. 2**). Similarly, PCC 7403<sup>T</sup> also clustered within the genus *Tumidithrix* with strong support in the 16S rRNA gene phylogenetic tree (**Fig. 3**). In addition to BACA0141<sup>T</sup> and PCC 7403<sup>T</sup>, the *Tumidithrix* clade also consisted of two misidentified strains, namely *Phormidium mucicola* IAM M-221 and *Pseudanabaena mucicola* NCT372 (**Fig. 3**). The 16S rRNA gene nucleotide similarity between PCC 7403<sup>T</sup> and the other strains of the genus *Tumidithrix* ranged from 99.0%–99.8%. Notably, its similarity to *Tumidithrix elongata* BACA0141<sup>T</sup> was 99.8%, higher than the delineation cutoff recommended by Yarza et al. (51). However, the pairwise ANI (average nucleotide identity) and AAI (average amino acid identity) measures between PCC 7403<sup>T</sup> and *Tumidithrix elongata* BACA0141<sup>T</sup> were 90.68% [95% CI: 90.46%–90.84%] and 92.33% [92.02%–92.64], respectively, whereas the digital DNA-DNA hybridization (dDDH; ggdc.dsmz.de, Formula 2) value was 41.50% [39.0%–44.0%]. These three overall genome relatedness indices (OGRI) values are clearly below their respective species delineation cutoffs, i.e. ANI < 95%, AAI < 96%, dDDH < 70%. The multilocus phylogenetic analysis, OGRI measures and the ability to perform nitrogen fixation under anaerobiosis confirms PCC
421

7403<sup>T</sup> to be a novel species within the genus *Tumidithrix* for which we propose the name

422

*Tumidithrix helvetica* sp. nov.

423

424

### 3.2.4 Analysis of the 16S-23S ITS rRNA

425

A single ITS rRNA operon (Location: CBDDRA010000060: 1719–2547) with both tRNA genes (tRNA<sup>Ile</sup> and tRNA<sup>Ala</sup>) was recovered from the genome assembly of PCC 7403<sup>T</sup>. The comparative sequence analysis of the D1-D1', V2, BoxB and V3 helices, the presence of tRNA<sup>Ile</sup> and tRNA<sup>Ala</sup>, and the position of the spacer region indicated that the ITS rRNA sequences of PCC 7403<sup>T</sup> and *Tumidithrix elongata* BACA0141<sup>T</sup> were of the same type (Supplementary Table S2), satisfying the necessary requirement for conducting the comparative analysis. The folded secondary structure of the D1-D1' helix of PCC 7403<sup>T</sup> consisted of a five bp basal clamp followed by eight nucleotides basal unilateral bulge opposed by a single nucleotide, an internal helix with three bilateral bulges and a terminal bilateral bulge (Fig. 7). This structure was similar to the folded secondary structure of BACA0141<sup>T</sup>; however, nucleotide differences were observed in the bilateral bulges of the internal helix. The folded secondary structure of the V2 helix of PCC 7403<sup>T</sup> consisted of a seven bp basal clamp followed by three bilateral bulges and a terminal bilateral bulge (Fig. 7). This structure was similar to the folded secondary structure of BACA0141<sup>T</sup>. However, the sizes of the terminal and two sub-terminal bilateral bulges were smaller than the corresponding bilateral bulges in PCC 7403<sup>T</sup>.

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434

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436

437

438

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440

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443

444

445

The folded secondary structure of the BoxB region of PCC 7403<sup>T</sup> was similar to the secondary structure of BACA0141<sup>T</sup>, and both of them consisted of a five bp basal clamp followed by a bilateral bulge, a single nucleotide unilateral bulge and a terminal bulge (Fig. 8). The base substitution of cytosine instead of uracil in PCC 7403<sup>T</sup> was the only observed difference between the two structures. Finally, the folded secondary structure of the V3 helix of PCC 7403<sup>T</sup> consisted of three bp basal stem followed by a bilateral bulge, a unilateral bulge and a
terminal bilateral bulge (Fig. 8). This structure was completely different from the folded
secondary structure of BACA0141T. The nucleotide dissimilarity between the ITS rRNA of
PCC 7403T and BACA0141T was 5.8%. According to the current accepted standards, the
nucleotide dissimilarity <3% and >7% indicate identical and different species, respectively,
whereas the dissimilarity values between 3%–7% is considered to be in range of ambiguity
(53). However, the OGRI measures between PCC 7403T and BACA0141T provides strong
evidence in their delineation at the species level (see section 3.2.3), indicating that the ITS
rRNA percent dissimilarity value of ~5.8% could be used as a threshold in the genus
*Tumidithrix*.

455

4. Taxonomic treatment

The taxonomic descriptions are based on the cytomorphological characters derived from this
study and the ultrastructural characters from Guglielmi and Cohen-Bazire (16).

4.1 Description of *Thalassoporum mexicanum* sp. nov.

*mexicanum* (me.xi.ca’num. N.L. neut. adj. *mexicanum*, belonging to Mexico, from where the
type strain was isolated).

Trichomes homocytic, short (3–10 cells), uniseriate, isopolar, not attenuated at the ends with
thin (but evident) “hyaline bridges”, always without sheath but sometimes with additional
cellular envelope external to the wall (extra layer). Trichomes (1.1)1.3–1.9(2.1) µm wide, with
cross-walls 3–6 times thicker than parietal peptidoglycan, and single central perforation.

Constrictions intensely deep, reducing the diameter of transverse septa to only 20–30% of the
trichome diameter. Transverse septa as circular disks and almost entirely situated around the
central perforation. Cells blue-green, usually barrel-shaped or cylindrical with rounded ends
and occasionally almost spherical (constricted and shorter than wide) with polar gas vesicles
and 6–10 peripherally arranged thylakoids. Spherical cells sometimes concatenate, forming
471 moniliform regions. Cells (1.4)1.9–3.7(4.1) µm long, usually 1.5× longer than wide, sometimes
472 slightly shorter than wide. Thylakoid membranes continuous around cells as concentric circles,
473 not interrupted at cell poles, always absent from centroplasm. Apical cells flat rounded or
474 conical rounded. Reproduction by intercellular trichome fragmentation and production of
475 hormogonia.

476 The type strain PCC 7367T (= BACA0810T) is epizoic and was isolated from a sample collected
477 from the surface of a snail shell in an intertidal zone in Puerto Penasco, Mexico. The genomic
478 DNA G + C content is 46.22 mol % (based on the genome sequence). The 16S rRNA gene and
479 the whole genome sequences are available under the GenBank/EMBL/DDBJ accession
480 numbers AM709633 and NC_019701, respectively.

481

482 4.2 Description of *Tumidithrix helvetica* sp. nov.

483 *helvetica* (hel.ve'ti.ca. L. fem. adj. *helvetica*, belonging to *Helvetia*, the Latin name of
484 Switzerland from where the type strain was isolated).

485 Trichomes homocytic, long, uniseriate, isopolar, straight or irregularly curved with thin (but
evident) “hyaline bridges”, always without sheath. Cross-walls 3–6 times thicker than parietal
486 peptidoglycan, and single central perforation. Constrictions intensely deep, reducing the
487 diameter of transverse septa to only 20–30% of the trichome diameter. Transverse partitions as
488 circular disks and almost entirely situated around the central perforation. Cells blue-green,
489 usually cylindrical, 2× longer than wide, with polar gas vesicles and 4–8 peripherally arranged
490 thylakoids. Centroplasm and region of cell poles always without thylakoids. Cell poles
491 occupied by a cytoplasmic area with ribosomes, carboxysomes and gas vesicles. Some
492 trichomes exhibit moniliform region resulting from the concatenation of the barrel-shaped
493 cells. Cylindrical cells 2.3–6.7 µm long and 1.3–2.4 µm wide, barrel-shaped cells 2.6–3.4 µm
494 long and 2.5–3.5 µm wide. Distinctly elongated cells frequently observed, 7.0–17.5 µm long
495
496

and 1.7–2.3 µm wide, sometimes irregularly swollen. Reproduction by intercellular trichome
fragmentation and formation of hormogonia.

497

498

The type strain PCC 7403T (= BACA0811T) was isolated from a sample collected from a
sphagnum bog near Kastanienbaum, Vierwaldstättersee, Switzerland. The genomic DNA G +
C content is 45.30 mol % (based on the genome sequence). The 16S rRNA gene and the whole
genome sequences are available under the GenBank/EMBL/DDBJ accession numbers
AB039019 and CBDDRA000000000, respectively.

499

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503

504

5. Conclusions

505

The present study introduces a novel species in each of the monotypic genera *Thalassoporum*
and *Tumidithrix* of the order *Pseudanabaenales* and provides further clarification in the
taxonomy of *Pseudanabaena* and *Pseudanabaena*-like taxa. The genetic diversity in both
genera, *Tumidithrix* and *Thalassoporum*, exceeded the morphological diversity, as evidenced
by the lack of unique morphological traits in the investigated strains. The analysis of the 16S
rRNA gene was inconclusive in species delineation in *Thalassoporum* and *Tumidithrix*.

506

507

508

509

510

511

Furthermore, while the analysis of the 16S-23S ITS rRNA region proved to be effective in
distinguishing the closely related species of *Thalassoporum*, it did not provide conclusive
evidence in delineating the closely related species of *Tumidithrix*. The currently accepted
nucleotide dissimilarity cutoff for the species delineation using the ITS rRNA region appears
to be conservative (because of the 4% ambiguity range) and may lead to an underestimation of
the actual species diversity within certain cyanobacterial genera. This limitation can be
addressed by incorporating genomic analyses into the taxonomic studies of cyanobacteria, as
demonstrated in this study. While the ITS rRNA region is widely used marker for
cyanobacterial species delineation, the currently accepted nucleotide dissimilarity cutoff
should be revisited and potentially redefined in accordance with the results emerging from

512

513

514

515

516

517

518

519

520
521 genomic data. Furthermore, the analysis of the NFGC in this study confirms its sporadic distribution and complex evolutionary trajectory within the order *Pseudanabaenales*. Finally, the presence of the BGC predicted to code for unknown glycolipid in the genome assembly of *Tumidithrix helvetica* PCC 7403T warrants further investigation.

525

526 Author contribution

527 **Aniket Saraf**: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & editing. **Boris Aleksovski**: Formal analysis, Writing – Original Draft Preparation, Writing – Review & Editing. **Eddy Blondet**: Methodology, Formal analysis, Investigation. **Svetislav Krstić**: Writing – Review & Editing. **Alexis Criscuolo**: Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & editing. **Muriel Gugger**: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & editing.

537

538 Acknowledgements

539 This work was supported by a Pasteur-Roux-Cantarini fellowship of Institut Pasteur. The Institut Pasteur is thanked for funding the PCC collection. For the purpose of open access, the author has applied a CC-BY public copyright licence to any Author Manuscript version arising from this submission. This work used the computational and storage services provided by the IT department at the Institut Pasteur. We also thank Prof. Aharon Oren for his help with the Latin etymology of both the scientific names. We are thankful for the comments of the anonymous reviewers.
546

547 **Conflict of interest disclosure**

548 The authors declare no conflict of interest.

549 **Funding information**

550 This research did not receive any specific grant from funding agencies in the public,

551 commercial, or not-for-profit sectors.

552

553

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731
<figure><img src="image_2.png" /><figcaption>**Figure 1:** Morphological attributes of *Thalassoporum mexicanum* PCC 7367T: (a) deeply constricted 3–10 celled trichomes with flat rounded or conical rounded apical ends; (b) cylindrical and barrel-shaped cells; (c) three celled hormogonium; (d) concatenated spherical cells resulting in moniliform region within a filament. Scale bars, 5 µm.</figcaption></figure>

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<figure><img src="image_3.png" /></figure>

740

741 **Figure 2:** Multilocus phylogenetic tree showing the position of *Thalassoporum mexicanum* PCC 7367T and *Tumidithrix helvetica* PCC 7403T. This ML phylogenetic tree includes 22 taxa belonging to the orders *Pseudanabaenales* and *Thermostichales*. Three *Gloeobacterales* taxa were used as an outgroup. Branch supports were assessed using Maximum Likelihood (ML)/Minimum Evolution (ME)/Maximum Parsimony (MP) bootstrap proportions; ‘-’ indicates branch support values lower than 70%. Bar, 0.5 amino acid substitutions per site.

747
<figure><img src="image_4.png" /><figcaption>**Figure 3:** 16S rRNA gene phylogenetic tree showing the position of *Thalassoporum mexicanum* PCC 7367T and *Tumidithrix helvetica* PCC 7403T. This ML phylogenetic tree includes 60 taxa belonging to the basal cyanobacterial lineages of *Pseudanabaenales*, *Thermostichales* and *Acaryochloridales*. Three *Gloeobacterales* taxa were used as an outgroup. Branch supports were assessed using Maximum Likelihood (ML)/Minimum Evolution (ME)/Maximum Parsimony (MP) bootstrap proportions; ‘-’ indicates branch support values lower than 70%. Bar, 0.05 nucleotide substitutions per site.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>V3</figcaption></figure>

756 **Figure 4:** Folded secondary structures of the D1-D1', BoxB and V3 helices of *Thalassoporum mexicanum* PCC 7367T and *Thalassoporum komarekii* TAU-MAC 1515T.
757
<figure><img src="image_6.png" /><figcaption>**Figure 5:** Morphological attributes of *Tumidithrix helvetica* PCC 7403T: (a–b) deeply constricted trichomes with cylindrical, elongated and slightly swollen cells; (c) barrel-shaped cells forming moniliform region; (d–e) trichomes with aberrant cell morphologies; (f–i) hormogonia. Scale bars, 5 µm.</figcaption></figure>

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<figure><img src="image_7.png" /><figcaption>**Figure 6:** Genetic organization of *nif* gene cluster (NFGC) of *Synechococcus* sp. JA-2-3B'a(2-13), *Leptolyngbya boryana* PCC 6306, *Tumidithrix helvetica* PCC 7403T and *Pseudanabaena* sp. PCC 6802. Assignment of types as in Chen et al. (58): Type A in yellow background and Type F in orange background. Genes encoding for structural components of nitrogenase are indicated in green whereas accessory *nif* genes are in blue, other unrelated genes are in orange and genes coding for unknown products are in grey.</figcaption></figure>

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<figure><img src="image_8.png" /><figcaption>**Figure 7:** Folded secondary structures of the D1-D1' and V2 helices of *Tumidithrix helvetica*</figcaption></figure>

774 PCC 7403<sup>T</sup> and *Tumidithrix elongata* BACA 0141<sup>T</sup>.

775

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<figure><img src="image_9.png" /><figcaption>**Figure 8:** Folded secondary structures of the BoxB and V3 helices of *Tumidithrix helvetica*</figcaption></figure>

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PCC 7403<sup>T</sup> and *Tumidithrix elongata* BACA 0141<sup>T</sup>.

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<table><caption>**Table 1:** Morphological, ultrastructural and ecological comparison between *Thalassoporum mexicanum* PCC 7367T and *Thalassoporum komarekii* TAU-MAC 1515T.</caption><thead><tr><th></th><th><em>Thalassoporum mexicanum</em> PCC 7367T</th><th><em>Thalassoporum komarekii</em> TAU-MAC 1515T</th></tr></thead><tbody><tr><td><strong>Trichome</strong></td><td>Homocytic, few celled (3–10 cells) uniseriate, isopolar and not attenuated at ends</td><td>Homocytic, few celled (3–10 cells) uniseriate, isopolar and not attenuated at ends</td></tr><tr><td><strong>Trichome width</strong></td><td>(1.1)1.3–1.9(2.1) µm</td><td>1.08–1.74 µm</td></tr><tr><td><strong>Sheath</strong></td><td>Absent but sometimes with additional cellular envelope</td><td>Absent</td></tr><tr><td><strong>Cell shape</strong></td><td>Barrel-shaped or cylindrical with rounded ends</td><td>Cylindrical with rounded ends</td></tr><tr><td><strong>Cell length</strong></td><td>(1.4)1.9–3.7(4.1) µm</td><td>1.3–3.24</td></tr><tr><td><strong>Moniliform region</strong></td><td>Observed, resulting from the concatenation of spherical cells</td><td>Not observed</td></tr><tr><td><strong>Constrictions at cross walls</strong></td><td>Deeply constricted</td><td>Deeply constricted</td></tr><tr><td><strong>Apical cell</strong></td><td>Flat rounded or conical rounded</td><td>Flat rounded or conical rounded</td></tr><tr><td><strong>Gas vesicles</strong></td><td>Present; polar</td><td>Present; polar</td></tr><tr><td><strong>Thylakoid number and arrangement</strong></td><td>6–10 peripherally arranged</td><td>5–6 peripherally arranged</td></tr><tr><td><strong>Reproduction</strong></td><td>Intercellular trichome fragmentation and hormogonia formation</td><td>NA</td></tr><tr><td><strong>Habitat</strong></td><td>Epizoic, marine, intertidal zone</td><td>Epizoic, marine, rocky sublittoral zone</td></tr><tr><td><strong>Location</strong></td><td>Puerto Penasco, Mexico</td><td>Porto Valitsa, Kassandra, Chalkidiki Peninsula, Greece</td></tr></tbody></table>

783 NA – Information not available

784

785
<table><caption>**Table 2:** Morphological, ultrastructural and ecological comparison between *Tumidithrix helvetica* PCC 7403T and *Tumidithrix elongata* BACA0141T.</caption><thead><tr><th></th><th><i>Tumidithrix helvetica</i> PCC 7403<sup>T</sup></th><th><i>Tumidithrix elongata</i> BACA0141<sup>T</sup></th></tr></thead><tbody><tr><td><strong>Trichome</strong></td><td>Homocytic, long, uniseriate, isopolar and straight or irregularly curved</td><td>Homocytic, long, uniseriate, isopolar, straight or irregularly waived</td></tr><tr><td><strong>Sheath</strong></td><td>Absent</td><td>Absent</td></tr><tr><td><strong>Cell shape</strong></td><td>Cylindrical, elongated and barrel-shaped</td><td>Cylindrical, elongated and barrel-shaped</td></tr><tr><td><strong>Cell Dimensions</strong></td><td>Cylindrical cells 2.3–6.7 µm long and 1.3–2.4 µm wide; barrel-shaped cells 2.6–3.4 µm long and 2.5–3.5 µm wide; elongated cells, 7.0–17.5 µm long and 1.7–2.3 µm wide</td><td>1.2–2.0 µm wide and 1.3–6.3 µm long; elongated cells up to 30 µm long.</td></tr><tr><td><strong>Moniliform region</strong></td><td>Observed, resulting from the concatenation of barrel-shaped cells</td><td>Not observed</td></tr><tr><td><strong>Constrictions at cross walls</strong></td><td>Deeply constricted</td><td>Deeply constricted</td></tr><tr><td><strong>Gas vesicles</strong></td><td>Present; polar</td><td>Present; polar</td></tr><tr><td><strong>Thylakoid number and arrangement</strong></td><td>4–8 peripherally arranged</td><td>Peripherally arranged</td></tr><tr><td><strong>Reproduction</strong></td><td>Intercellular trichome fragmentation and hormogonia formation</td><td>Hormogonia formation</td></tr><tr><td><strong>Habitat</strong></td><td>Sphagnum bog</td><td>Freshwater</td></tr><tr><td><strong>Location</strong></td><td>Kastanienbaum, Vierwaldstättersee, Switzerland</td><td>Lagoa do Paul, Pico Island (Azores), Portugal</td></tr></tbody></table>