bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 Characterization of isolated from thermal muds of Balaruc- 3 4 Les-Bains (France) and description of a new genus and Pseudo- 5 6 chroococcus couteii 7 8 Duval C.1, Hamlaoui S.1, Piquet B.2, Toutirais G.2, Yéprémian C.1, Reinhardt A.3, Duperron S.1, Marie B.1, 9 10 Demay J.1, Bernard C.1* 11 12 13 1 UMR7245 MCAM MNHN-CNRS, Muséum National d’Histoire Naturelle, CP 39, 12 rue Buffon, F- 14 15 75231 Paris Cedex 05, France. 16 17 2 Electron Microscopy Platform, Muséum National d’Histoire Naturelle, CP 39, 12 rue Buffon, F-75231 18 19 Paris Cedex 05, France. 20 3 Thermes de Balaruc-Les-Bains, 1 rue du Mont Saint-Clair BP 45, 34540 Balaruc-Les-Bains ; 21 22 [email protected] 23 24 25 * Correspondance: [email protected]; Tel.: +33 1 40 79 31 83 26 27 28 29 ABSTRACT 30 31 Cyanobacteria are able to synthesize a high diversity of natural compounds that account for their success in 32 33 the colonization of a variety of ecological niches. Many of them have beneficial properties. The mud from 34 the thermal baths of Balaruc-Les-Bains, one of the oldest thermal baths in France, has long been 35 36 recognized as a healing treatment for arthro-rheumatic diseases. To characterize the cyanobacteria living in 37 38 these muds and the metabolites they potentially produce, several strains were isolated from the water 39 40 column and biofilms of the retention basin and analyzed using a polyphasic approach. Morphological, 41 ultrastructural and molecular (16S rRNA gene and 16S-23S ITS region sequencing) methods were 42 43 employed to identify nine cyanobacterial strains belonging to the orders , Synechococcales, 44 45 and . The combination of morphological and genetic characteristics supported the 46 description of a new genus and species with the type species as Pseudo-chroococcus couteii. The high 47 48 taxonomic diversity in the muds of the thermal baths of Balaruc-Les-Bains along with literature reports of 49 50 the potential for bioactive metabolite synthesis of these taxa allowed us to hypothesize that some of the 51 52 metabolites produced by these strains could contribute to the therapeutic properties of the muds from 53 Thermes de Balaruc-Les-Bains. 54 55 56 57 KEYWORDS 58 59 cyanobacteria, thermal mud, , polyphasic approach, morphology, ultrastructure. 60

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. INTRODUCTION 1 2 Cyanobacteria belong to an ancient group of photosynthetic prokaryotes presenting a broad range of 3 4 cellular strategies, physiological capacities, and adaptations that support their colonization of diverse 5 6 environments worldwide. Cyanobacteria can even exist in extreme habitats and are able to settle in diverse 7 biotopes such as hot springs. They are also known for their production of natural bioactive compounds 8 9 (Demay et al. 2019), including some potent toxins (microcystins, anatoxins, ) (Bernard et al. 10 11 2017). Some of these metabolites have been used for applications in the biotechnology and pharmaceutical 12 13 fields, which has created an increased interest in the search for new isolates of cyanobacteria. Both the 14 chemical diversity and the related bioactivity must be considered when investigating the application 15 16 potential of natural products. Demay et al. (2019) concluded that among the 300 different recognized 17 18 genera of cyanobacteria (referenced by the taxonomy published by Komárek et al. in 2014), 90 have been 19 20 reported to produce bioactive metabolites. A few taxa are known to be prolific producers of a large set of 21 metabolites. For example the genus Lyngbya-Moorea, produces 85 families of the 260 metabolites isolated 22 23 so far. The majority of species have not been tested, however, and the potential for the discovery of useful 24 25 natural molecules and new biosynthetic pathways from cyanobacteria remains considerable and needs to be 26 explored. 27 28 Thermes de Balaruc-Les-Bains is one of the oldest thermal centers in France and therapeutic application of 29 30 the mud, whose beneficial effects have been documented since the end of the 19th century, was recognized 31 32 by the French Health System as a healing treatment for arthro-rheumatic diseases. The beneficial mud is 33 obtained by a process of maturation in which the mud is allowed to settle naturally from a combination of 34 35 hot spring water and domestic rinse water from mud treatments in a large tank. Two types of biofilms were 36 37 identified within the maturation basin, one at the surface of the mud and the other on the walls of the basin. 38 39 Other photosynthetic microorganisms were also described in the water column of the basin. Only two 40 experimental studies have been carried out on mud from Thermes de Balaruc-Les-Bains. The maturation of 41 42 silt from Thau Pond in Balaruc-Les-Bains was monitored for a few months in 1983, and algal development 43 44 was observed ten days after formation of a mud mesocosm comprised of Phormidium and Oscillatoria 45 Cyanobacteria, and some Diatomophyceae (Baudinat 1986). Repeated experiments in 1984 gave similar 46 47 results except that algal development occurred later, after one month (Baudinat 1986). The second study 48 49 was carried out in 1987 by Dupuis in experimental mesocosms for thermal mud maturation in the 50 51 laboratory. In the absence of mud, there was no algal development in water from Balaruc-Les-Bains 52 despite the addition of nutrients. In the mud mesocosms at seven weeks after the experiment started, very 53 54 thick cyanobacterial mats were observed on walls and bottom, dominated by Phormidium africanum, P. 55 56 autumnale, Lyngbya sp., Chroococcus minor and Anabaena sp. These two studies illustrated the 57 58 importance of cyanobacteria as major actors in the colonization of the mud of Thermes de Balaruc-Les- 59 Bains (Baudinat 1986 & Dupuis 1987). 60

2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. In the present study, we analyzed the diversity of cyanobacteria isolated from the mud of Thermes de 1 2 Balaruc-Les-Bains. Nine clonal but non-axenic strains of cyanobacteria were found and their taxonomy 3 4 was verified before genomic and metabolomic analysis and bioactivities studies on their natural products 5 were conducted. The diversity of the strains was quite high, including members of four orders, 6 7 Chroococcales, Synechococcales, Oscillatoriales and Nostocales. 8 9 10 11 MATERIALS AND METHODS 12 13 Sampling 14 Samples for strain isolation were collected twice a month from April 28 to October 13, 2014 (Table 1) 15 16 from the thermal basins of Balaruc-Les-Bains (43°26'44.0"N 3°40'29.6"E). 17 18 The 500 m3 maturation basin (Figure 1a, c and d) received the muddy water from the rinsing of the patients. 19 20 The slurry was a mixture of water from the thermal springs along with domestic hot water containing the 21 mud particles, which settle to the bottom (Figure 1b and c). 22 23 The water in the retention basin was stirred by the continuous influx of rinse water, and the level was kept 24 25 above the mud by draining excess water to the outside to prevent overflow (Figure 1d). The proper 26 temperature and light levels and nutrient inputs were maintained to ensure that the mud maturation process 27 28 was optimal. Three types of sampling were carried out from the maturation basin: (i) from the biofilm 29 30 covering the mud, (ii) from the epilithic biofilm on the walls of the basin where algae development was 31 32 more extensive and (iii) from the water surface using a 20-μm membrane filter. 33 34 35 Cyanobacterial culture conditions and strain isolation 36 37 Samples were inoculated on solid medium (5 or 10 g∙L−1 agar) Z8 and Z8-salts (Rippka 1988). Isolations 38 39 were carried out by repeated transfers (at least three times) of single cells or filaments on solid or liquid 40 media under an inverted microscope (Nikon ECLIPSE TS100). Viable clones were then cultured in 25 cm2 41 42 culture flasks (Nunc, Roskilde, Denmark) containing 10 mL of Z8. Cyanobacterial strains were maintained 43 44 in the Paris Museum Collection (PMC) at 25°C, using daylight fluorescent tubes providing an irradiance of 45 12 μmol photons∙m−2∙s−1, with a photoperiod of 16h light/8h dark. Isolated strains and cultures were all 46 47 monoclonal and non-axenic. 48 49 50 51 Morphological analyses 52 Morphological analyses of cyanobacterial strains were carried out using an Axio ImagerM2 Zeiss 53 54 microscope. Photographs of the specimens were taken with an AxioCam MRc digital camera coupled to 55 56 the microscope and images were processed using the ZEN software (Zeiss). The cell and filament width 57 58 and length, morphology, color and motility were determined. Strains were identified morphologically using 59 the updated taxonomic literature (Komárek and Anagnostidis 2005; Komárek and Anagnostidis 2007; 60 Komárek and Johansen 2015).

3 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 Pigment analysis 3 4 Pigment analysis was performed on each strain in triplicate. Ten mL aliquots of culture in exponential 5 growth were filtered through Whatman GF/F filters (Ø 0.7 µm) and freeze-dried. The lipophilic 6 7 carotenoids and chlorophylls were extracted, analyzed and quantified by HPLC-DAD as described (Ras et 8 9 al., 2008). The water soluble phycobiliproteins were analyzed following a Standard Operating Procedure 10 11 (Yéprémian et al. 2017). 12 13 14 Ultrastructural analyses 15 16 Cyanobacterial strains were imaged by scanning electron microscopy (SEM) and transmission electron 17 18 microscopy (TEM) as described by Parveen et al. (2013), with modifications. Cells or filaments harvested 19 by centrifugation from a growing culture were fixed with 2% (v/v) glutaraldehyde, 2% (v/v) formaldehyde, 20 21 0.18 M sucrose, and 0.1% picric acid in 0.1 M Sorensen phosphate buffer (SPB; pH 7.4) for 1 h, at room 22 23 temperature. The specimens were then washed three times with SPB and post-fixed with 1% osmium 24 tetroxide for 1 h. Afterwards, they were washed with distilled water before being dehydrated in a 25 26 microscopy-grade ethanol series (30%, 50%, 70%, 90% and 100%), with agitation and centrifugation. For 27 28 SEM, the samples were pipetted onto glass coverslips on SEM stubs and air dried. They were coated with 29 30 platinum (Leica EM ACE600 coater) and examined using a scanning electron microscope (Hitachi 31 SU3500, Japan). For TEM, the samples were embedded in epon resin and sectioned at 0.5 μm using an 32 33 ultra-microtome (Reichert-Jung Ultracut) with a diamond knife and transferred onto 150-mesh copper 34 35 grids. The prepared sample grids were stained with 2% uranyl acetate in 50% ethanol for 15 min and 36 37 washed three times in 50% ethanol and twice in distilled water. The copper grids were then dried, 38 examined with a transmission electron microscope (Hitachi HT-7700, Japan), and photographed with a 39 40 digital camera (Hamamatsu, Japan). 41 42 43 Molecular and phylogenetic analyses 44 45 DNA was extracted from cyanobacterial strains using the ZymoBIOMICS DNA mini kit (Zymo Research, 46 47 CA) following the manufacturer’s protocol. Cells were lysed using a Qiagen bead mill for 6 min at 48 49 maximum speed (30 Hz, 1800 oscillations per minute). The amplification of the 16S rRNA-encoding gene 50 and the 16S-23S internal transcribed spacer (ITS) region was done with the primers and PCR programs 51 52 described in Cellamare et al. (2018) and Gama et al. (2019), respectively. All PCR reactions were carried 53 54 out using a GeneTouch thermocycler (Bioer Technology, Hangzhou, China). The extracted and purified 55 PCR products were then sequenced by Genoscreen (Lille, France). Sequences were assembled and 56 57 corrected using the MEGA version 7.0 software (Kumar et al. 2016) and aligned using MAFFT online 58 59 service (Katoh, et al. 2019). For the 16S rRNA gene, partial sequences with a minimum of 1398 base pairs 60 (bp) from the complete gene sequence (1550 bp) were obtained. For the 16S-23S ITS, partial sequences

4 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. were obtained with a minimum of 174 bp. Phylogenetic analyses were performed according to three 1 2 methods: maximum parsimony (MP), neighbor joining (NJ), and maximum likelihood (ML) using the 3 4 MEGA version 7 software with an equal-to-1000 iterations. 5 For 16S rRNA gene phylogeny, an overall alignment (n = 129 sequences) was generated, including the 6 7 newly produced sequences and reference sequences available in GenBank representing Oscillatoriales, 8 9 Synechococcales, Nostocales and Chroococcales. The selected sequences were all longer than 1200 bp. 10 11 Gloeobacter violaceus PCC 7421 was chosen as outgroup. A cut-off value of 95% of 16S rRNA gene 12 sequence identity was used for genus definition (Komárek 2010). 13 14 For the Chroococcus-like strain, the 16S-23S ITS phylogeny was performed with representative strains 15 16 from Chroococcus-like, Inacoccus, Cryptococcum, Gloeocapsa (syn. Limnococcus) and Chroococcus, 17 18 whose sequences are available in Genbank and (Gama et al. 2019). An overall alignment (n = 21) was 19 generated with the selected sequences, all longer than 1494 bp. Microcystis aeruginosa PMC 728.11 was 20 21 chosen as an outgroup. The generated 16S-23S ITS sequences were used for determination of secondary 22 23 structure. The conserved regions (D1–D1’) were analyzed using the Mfold WebServer (Zuker 2003). 24 Default settings were used, except for the structure drawing mode where the natural angle was selected. 25 26 Transfer RNA genes were found using tRNAscan-SE 2.0 (Lowe and Chan 2016). The nucleotide 27 28 sequences reported in this study have been deposited in the National Center for Biotechnology Information 29 30 database. GenBank accession numbers are listed in the Supplementary table (Table S1). 31 32 33 RESULTS AND DISCUSSION 34 35 Nine clonal strains of cyanobacteria, living in the mud maturation basin of the Thermes de Balaruc-Les- 36 37 Bains, were isolated (Table 1). These organisms were representative of the algal communities in (Baudinat 38 39 1986; Dupuis 1987). Dupuis (1987) showed that Phormidium was the most abundant genus in muds of 40 Balaruc-Les-Bains with the species Phormidium africanum as dominant and P. autumnale, Lyngbya sp. 41 42 and Chroococcus as accompanying species. Baudinat (1986), also showed that the cyanobacteria were 43 44 dominant, mainly represented by Phormidium and Oscillatoria. A preliminary study done in 2014 during 45 the mud maturation period showed that the cyanobacterium, Planktothricoides raciborskii, was also quite 46 47 dominant (unpublished data). Other taxa such as Laspinema sp., boryana and sp. 48 49 were observed. The dominance of Laspinema sp. was observed on one sampling date, corresponding to a 50 51 slight increase in salinity of the thermal water. Other cyanobacterial taxa were observed such as 52 sp. in the mud and Aliinostoc sp., Chroococcus-like and Microcoleus vaginatus on the walls of the 53 54 maturation basin. As Planktothricoides raciborskii is morphologically very similar to Phormidium, we 55 56 concluded that the dominance of the genus Phormidium has been stable since the 1980s. 57 58 To characterize the nine cyanobacterial isolates, we used morphological, ultrastructural and 16S rRNA and 59 16S-23S internal transcribed spacer (ITS) gene sequence analyses. Thus, eight cyanobacteria were firmly 60 identified at the genus or species level, while one remained unidentified (PMC 885.14). The phylogenetic

5 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. trees based on the 16S rRNA gene sequences shared well-supported nodes at the order level regardless of 1 2 whether the method used was MP, NJ or ML (see the ML tree, Fig. 2, and the three consensus phylogenies, 3 4 Fig. S2). Some isolates were clearly identified at the species level, while others were only assigned to the 5 genus level because of the large species diversity within the given genus. 6 7 The nine new strains belonged to the orders Chroococcales: Pseudo-chroococcus couteii (PMC 885.14); 8 9 Synechococcales: Leptolyngbya boryana (PMC 883.14); Oscillatoriales: Planktothricoides raciborskii 10 11 (PMC 877.14), Laspinema sp. (PMC 878.14), Microcoleus vaginatus (PMC 879.14), Lyngbya martensiana 12 (PMC 880.14) and Nostocales: Nostoc sp. (PMC 881.14), Aliinostoc sp. (PMC 882.14), Calothrix sp (PMC 13 14 884.14). Identification numbers and habitats of the strains are given in Table 1. 15 16 17 18 Characterization of strains belonging to the Chroococcales 19 Among the nine studied strains, one unidentified strain displayed a Chroococcus-like morphology (PMC 20 21 885.14) and belonged to the Chroococcales; but, the 16S rRNA sequence diverged from known 22 23 Chroococcus sequences by more than 5%, supporting the idea that it was a new genus. 24 25 26 Pseudo-chroococcus couteii (C. Duval, S. Hamlaoui, C. Bernard gen. nov., sp. nov., 2020) (PMC 885.14) 27 28 Pseudo-chroococcus couteii (PMC 885.14) was isolated from an epilithic biofilm in the mud maturation 29 30 basin of Thermes de Balaruc-Les-Bains (Table 1). Examination of samples in the field showed single cells 31 or small colonies of 2-8 cells surrounded by dense, colorless and slightly lamellate mucilaginous envelopes 32 33 (Fig. 3). Under both natural and laboratory conditions, cells were spherical or hemispherical 14-18 µm in 34 35 diameter with pale-green, blue-green or grey homogeneous to granulate cell contents (Fig. 3). Cell division 36 37 occurred by binary fission in three or more planes or irregularly in old colonies. These morphological 38 features are typical of the genus Chroococcus. TEM ultrastructure imaging showed a dense mucilaginous 39 40 layer surrounding four grouped cells (Fig. 3). Thylakoids were fasciculate, as found in Chroococcus 41 42 species. 43 The 16S rRNA gene sequence analyses showed that the Pseudo-chroococcus couteii strain PMC 885.14 44 45 had 93% and 92% sequence identity with Inacoccus and Cryptococcum gen. nov. strains respectively (data 46 47 not shown). The 16S rRNA sequence data also revealed the polyphyletic nature of the genus Chroococcus. 48 49 The ML phylogenetic tree of the 16S rRNA and the 16S-23S ITS gene sequences (Fig. 4 and Fig. 5) of 50 Chroococcus strains produced three main clusters, Chroococcus-like, Inacoccus/Cryptococcum and 51 52 Limnococcus/Gleocapsa, separated from the Chroococcus sensu stricto cluster. The first cluster was 53 54 mainly composed of three strains of Inacoccus carmineus from concrete of Santa Virgínia Park in Brazil 55 and two strains of Cryptococcum from thermal springs and soil. The second cluster grouped Limnococcus 56 57 limneticus and Gleocapsa sp. 16S rRNA gene sequences. The third Chroococcus-like cluster consisted of 58 59 two sub-clusters: one composed of three marine strains isolated from surface sediments in New Zealand 60

6 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. and Hawaii, and one composed of PMC 885.14 (this study) and four freshwater strains from China. The 1 2 two sub-clusters were well-supported (100% bootstrap support). 3 4 Substantial differences were observed among the secondary ITS structures (D1-D1’ helices) of the three 5 genera previously mentioned (Fig. 6). The pattern and the number of nucleotides (nt) in the D1-D1’ region 6 7 of Chroococcus sensu stricto (58 nt), Inacoccus carmineus (63-64 nt), Cryptococcum (56-59 nt) and 8 9 Chroococcus-like (48-49 nt) were different. The pattern of the terminal portion of Pseudo-chroococcus 10 11 couteii strain (PMC 885.14) from Thermes de Balaruc-les-Bains was different from that of freshwater 12 strains from China (YW-2019b strain TP20176.5 clone 0.1 and CHA TP20176.4 clone 0.6), with a single 13 14 loop of 18 nt for Pseudo-chroococcus couteii and two loops of 4 nt and 9 nt for the Chinese strains (Fig. 6). 15 16 A unilateral bulge of 7 nt (CAAUCC) was present in the three strains of the Chroococcus-like cluster. 17 18 To the best of our knowledge, only four studies to date have examined phylogenetic relationships (16S 19 rRNA) within the genus Chroococcus (Komărkovă et al. 2010; Roldán et al. 2013; Wood et al. 2017; 20 21 Gamma et al. 2019). The strains studied were all morphologically similar to Chroococcus species in cell 22 23 shape and organization (Table S2). However, they differed by the color of the sheaths or mucilage (e.g., the 24 mucilage of Chroococcus was usually hyaline or yellowish whereas that of Limnococcus was described as 25 26 red or brown) (Gama et al. 2019; Table S2). Wood et al. (2017) stated that within the 51 Chroococcus-like 27 28 16S rRNA sequences, only five were obtained from isolates, limiting phylogenetic investigations. 29 30 However, this study included the new 16S rRNA sequences available in GenBank and clearly 31 demonstrated that the Chroococcus-like cluster was well-supported and should be described as a new 32 33 genus. 34 35 36 37 Pseudo-chroococcus couteii by C. Duval, S. Hamlaoui, C. Bernard gen. nov., sp. nov. 38 Diagnosis: Solitary cells or small colonies of 2, 4 or 8 cells surrounded by dense, colorless and slightly 39 40 lamellate mucilaginous envelopes. Cells, spherical or hemispherical 14-18 µm in diameter with pale-green, 41 42 blue-green or grey homogeneous to granulate cell contents. Cell division occurring by binary fission in 43 44 three or more planes, or irregularly in old colonies. 45 Holotype: A formaldehyde-fixed, cryopreserved sample of the strain PMC 885.14 was deposited at the 46 47 Paris Museum Collection (PMC), Paris, France. 48 49 Type strain: A live culture was deposited at the Paris Museum Collection (PMC) as PMC 885.14, with 50 GenBank accession no. MT985488 for the 16S-23S rRNA gene sequence. 51 52 Type locality: epilithic biofilm, retention basin of the thermal springs at Balaruc-Les-Bains, France (GPS: 53 54 43°26'44.0"N 3°40'29.6"E). 55 56 Etymology: the name of the genus, Pseudo-chroococcus, refers to the cyanobacterium morphology 57 (colonial) like Chroococcus sensu stricto, but, clearly phylogenetically separate in two different clades; 58 59 couteii = named in honor of Prof. Alain Couté, a French phycologist at the National Museum of Natural 60 History in France.

7 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 Characterization of strains belonging to the Synechococcales 3 4 Among the nine studied strains, one strain Leptolyngbya boryana (PMC 883.14) belonged to the 5 Synechococcales. 6 7 8 9 Leptolyngbya boryana (Anagnostidis et Komárek, 1988) 10 11 Leptolyngbya boryana (PMC 883.14) was isolated from mats covering the surface of the basin of Thermes 12 de Balaruc-Les-Bains (Fig. 1, Table 1). Examination of samples in the field showed a pale-green, diffluent 13 14 thallus forming mats (Fig. 7). Under laboratory conditions, the filaments were straight to slightly curved 15 16 and densely entangled with thin, firm, colorless sheaths. Trichomes were pale to bright blue-green, 17 18 cylindrical, unbranched, thin, < 3 µm wide and strongly constricted at the ungranulated cross-walls (Fig. 19 7), motile (hormogonia) or immotile, or with indistinct trembling. Cells were moniliform, shorter than 20 21 wide, 1.5-2.4 μm wide and 0.9-2.0 µm long with a length:width ratio of 0.76 and well-separated from one 22 23 another by cross-walls. Cell contents were pale blue-green and homogeneous without gas vacuoles. Apical 24 cells were rounded without calyptra (Fig. 7). A sheath with a thin transparent zone covered the trichome 25 26 and was clearly visible by TEM. The thylakoids were parietally arranged, concentric and parallel to the cell 27 28 wall (Fig. 7). 29 30 Results based on 16S rRNA gene sequence homology (BLAST), showed that the Leptolyngbya strain 31 (PMC 883.14) shared 100% sequence identity with other Leptolyngbya strains (data not shown). This result 32 33 was confirmed by the phylogenetic tree (Fig. 8) where the strain PMC 883.14 was grouped in a well- 34 35 supported clade including the Leptolyngbya boryana, cluster with one Leptolyngbya foveolarum sequence 36 37 (X84808.1) and one Leptolyngbya boryanum sequence (X84810.1). The cluster was mainly comprised of 38 strains isolated from benthic freshwater environments. 39 40 The morphological features of the Leptolyngbya boryana PMC 883.14 strain were very similar to other 41 42 Leptolyngbya species belonging to the same phylogenetic cluster (Table S3) which made it difficult to 43 distinguish. In Komárek and Anagnostidis (2005), Leptolyngbya foveolarum was described with the 44 45 following characteristics: variously curved filaments, sometimes straight and parallel or tangled, rarely 46 47 pseudo-branched; trichomes pale to bright blue-green1-2 µm wide; isodiametric cells, rarely longer than 48 49 wide 0.8-2.2 x 2.5 µm. Leptolyngbya boryana was described as follows: curved densely tangled filaments, 50 sometimes pseudo-branched, pale blue-green to colorless, 1.3-2 (3) µm wide, cells more or less 51 52 isodiametric, somewhat shorter or longer than wide in the main filament 0.6-2 (2.5) x 1.3-3 µm. In other 53 54 phylogenetic studies, several Leptolyngbya species such as L. foveolarum, L. tenerrima, and L. angustata 55 were considered as synonyms of L. boryana (Cuzman et al. 2010). As the genus Leptolyngbya was defined 56 57 based on strains with very thin and simple trichomes (0.5-3.5 μm wide), sheaths present or facultative, 58 59 without gas vesicles and peripherally arranged thylakoids (Komárek and Anagnostidis 2005; Komárek and 60 Johansen 2015), these morphological features were deemed not sufficiently discriminant to ensure a proper

8 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. identification; therefore, we consider the strain PMC 883.14 as Leptolyngbya boryana based on the former 1 2 description of this species within the Leptolyngbya 16S rRNA gene sequences cluster. 3 4 5 Characterization of strains belonging to the Oscillatoriales 6 7 Among the nine studied strains, four strains belonged to the Oscillatoriales: Planktothricoides raciborskii 8 9 (PMC 877.14), Laspinema sp. (PMC 878.14), Microcoleus vaginatus (PMC 879.14) and Lyngbya 10 11 martensiana (PMC 880.14). 12 13 14 Planktothricoides raciborskii (Wołosz) (Suda et M. M. Watanabe, 2002) 15 16 Planktothricoides raciborskii (PMC 877.14) was isolated from mats covering the mud of the basin (Fig. 1, 17 18 Table 1). Observations of field samples showed pale-green to brown diffluent thalli (Fig. 10). Trichomes 19 were solitary, pale green to brown, isopolar or heteropolar, cylindrical, variously elongated (100-450 µm). 20 21 Sheaths were seen occasionally, and were very thin and colorless, with limited motility (gliding). 22 23 Trichomes were attenuated towards extremities, constricted or no constricted and sometimes slightly bent 24 near the apex (Fig. 10). Cells were cylindrical, shorter than wide, 5-7.3 µm wide and 3.3-5.4 µm long, with 25 26 a length:width ratio of 0.7. Apical cells were rounded, conical, more or less tapered, sometimes bent but 27 28 not sharply pointed, without calyptra (Fig. 10; Table S4). Cell content was granular with cyanophycin 29 30 granules and carboxysomes (polyhedral or dense bodies; Fig. 10). These proteinaceous micro- 31 compartments play a major role in the carbon-fixation process by increasing the CO concentration around 32 2 33 RuBisCO, thus promoting carboxylation at the expense of oxygenation. Sheaths were generally thin and 34 35 some trichomes were associated with under our laboratory culture conditions. Cross-walls were 36 37 clearly visible with TEM (Fig. 10) and the thylakoids were arranged radially in cross sections of the 38 trichomes. 39 40 The 16S rRNA gene sequence comparisons (BLAST) showed that the PMC 877.14 strain shared ≥ 99% 41 42 sequence identity with other Planktothricoides strains (data not shown). The phylogenetic tree of the 43 44 sequences of Planktothricoides produced two main clusters (Fig. 9). The first cluster was primarily 45 composed of strains isolated from freshwater habitats, the one from Thermes de Balaruc-Les-Bains, one 46 47 strain from Thailand, one from Australia, one from Japan and one from Singapore. The second cluster 48 49 grouped the strains Candidatus ‘Planktothricoides niger’ and Candidatus ‘Planktothricoides rosea’ from 50 periphyton mats of tropical marine mangroves in Guadeloupe (Guidi-Rontani et al. 2014). The two species, 51 52 Candidatus ‘Planktothricoides niger’ and Candidatus ‘Planktothricoides rosea’, have been distinguished by 53 54 their phycoerythrin pigment content. The Planktothricoides strain from Thermes de Balaruc-Les-Bains 55 56 (PMC 877.14) contained two dominant phycobilin pigments, phycoerythrin (PE) and phycocyanin (PC), 57 with a PC:PE ratio = 1 (Fig. S1, Table S4). As the color of trichomes differs between field samples and 58 59 laboratory cultures, we suggest that this strain can either undergo complementary chromatic adaptation 60 (Grossman, 2003) or modify its PC:PE ratio depending on light wavelength or penetration through the

9 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. biomass. This characteristic is clearly different from the type description of Planktothricoides raciborskii 1 2 (Wołosz.) Suda and M. M. Watanabe (Suda et al., 2002; Komárek et al. 2004), where phycoerythrin was 3 4 described as absent and complementary chromatic adaptation was not observed. The presence of PE should 5 not be considered as sufficient to assign the strain PMC 877.14 to either Planktothricoides niger or 6 7 Planktothricoides rosea, and we considered the phylogenetic data strong enough to label the PMC 877.14 8 9 strain as Planktothricoides raciborskii. 10 11 12 Laspinema sp. (F. Heidari & T. Hauer, 2018) 13 14 The Laspinema strain (PMC 878.14) was isolated from the mud covering the maturation basin of Thermes 15 16 de Balaruc-Les-Bains. Examination of field samples showed a pale green to brown diffluent thallus (Fig. 17 18 11). In culture, the strain showed green or blackish green thallus formed mats and its trichomes were bright 19 blue-green often gradually attenuated at the ends, long, straight, not constricted at the finely granulated 20 21 cross-walls, bent, hooked and intensely motile (Fig. 11). Cells were always shorter than wide, 3.8-4.8 µm 22 23 wide, 2.3-3.3 µm long with a length:width ratio of 0.7. Sheaths were generally absent (Fig. 11). The apical 24 cells were obtusely-rounded to rounded-conical without calyptra, and often with thickened outer cell walls 25 26 (Fig. 11). 27 28 The thylakoids visible in cross sections of the trichomes were radially arranged with several 29 30 interthylakoidal spaces (Fig. 11). Other components such as numerous small gas vesicles or aerotopes, 31 which promote flotation of the filaments, carboxysomes, cyanophycin granules (nitrogen reserves), 32 33 glycogen, lipids, or poly-β-hydroxybutyrate acting as carbon and energy sources, and polyphosphate 34 35 granules (phosphorus reserves) were observed within the cells 36 37 The 16S rRNA gene sequence analysis (BLAST) showed that the PMC 878.14 strain shared ≥99% 38 sequence identity with other Laspinema sp. strains (data not shown). The phylogenetic tree of Laspinema 39 40 strains had one robust cluster (Fig. 9) composed of several species including L. lumbricale, L. thermale and 41 42 L. etoshii. Comparison of the morphological characteristics of the different species (Table S5) did not 43 44 allow us to assign a species name to the studied strain. Thus, strain PMC 878.14 was named Laspinema sp. 45 based on morphology (Table S5) and phylogenetics (Fig. 9). 46 47 48 49 Microcoleus vaginatus (Gomont ex Gomont 1892) 50 Microcoleus vaginatus (PMC 879.14) was isolated from the wall of the retention basin. The field samples 51 52 showed a dense green or blackish green thallus (Fig. 12). In culture, the thallus was green or blackish 53 54 green, forming mats and showing motility (gliding). Trichomes were often gradually attenuated at the ends, 55 56 with no constrictions at the cross-walls (Fig. 12). The cell wall envelope consisted of a cytoplasmic 57 membrane bounded by relatively thin wall layers. The apical cells were rounded with rounded or truncated 58 59 calyptra, often with thickened outer cell walls (Fig. 12). Cells were always shorter than wide, 4.7-6.2 µm 60 wide by 2.4-4.8 µm long, with a length:width ratio of 0.7 (Table S6).The thylakoids formed fascicle-like

10 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. aggregations arranged irregularly in the cells. The cells contained several large cyanophycin granules, 1 2 some carboxysomes and numerous small gas vesicles or aerotopes (Fig. 12). 3 4 The 16S rRNA gene sequence analysis (BLAST) showed that PMC 879.14 shared ≥98% sequence identity 5 with other Microcoleus vaginatus strains (data not shown). The phylogenetic tree placed the sequence in a 6 7 well-grouped cluster containing numerous M. vaginatus sequences (Fig. 9). Some sequences belonging to 8 9 Oscillatoria amoena and O. nigro-viridis were also grouped in this cluster, probably because they were 10 11 assigned before the taxonomic revision of this group (Strunecky et al. 2013). According to phylogeny and 12 following the recent taxonomic revision, the strain PMC 879.14 was assigned to Microcoleus vaginatus. 13 14 15 16 Lyngbya martensiana (Meneghini ex Gomont 1892) 17 18 Lyngbya martensiana (PMC 880.14) was isolated from the epilithic biofilm of the retention basin of 19 Thermes de Balaruc-Les-Bains. Field examinations and laboratory cultures showed bright-green thalli 20 21 composed of tangled filaments, usually arranged in parallel (Fig. 13). Filaments were long, straight or 22 23 variously flexuous with thick colorless sheaths. Trichomes were cylindrical and not constricted at the 24 cross-walls. The granular cell contents were pale blue-green. Cells were discoid, shorter than wide, 4.8-6.6 25 26 (5.3±0.47) µm wide, 1.2-2.1 (1.6±0.2) µm long with a length:width ratio of 0.3. Apical cells were widely 27 28 rounded, hemispherical or depressed-hemispherical without calyptra (Fig. 13). The thylakoids formed 29 30 fascicle-like aggregations arranged irregularly in the cells and situated near walls and cross-walls. 31 Numerous pores penetrating the cross-walls between the cells were clearly visible (Fig. 13). Gas vesicles 32 33 occurred throughout the cells, which also contained reserve granules and some carboxysomes (Fig. 13). 34 35 The 16S rRNA gene sequence analysis showed that PMC 880.14 shared ≥96% sequence identity with other 36 37 Lyngbya martensiana strains (data not shown). The phylogenetic tree of the sequences of Lyngbya 38 martensiana strains produced one well-grouped cluster (Fig. 9). The morphological characteristics (Table 39 40 S7) and the well-grouped Lyngbya martensiana cluster allowed us to name the PMC 880.14 strain as 41 42 Lyngbya martensiana (Gomont M. 1892). 43 44 45 Characterization of strains belonging to the Order Nostocales 46 47 Among the nine studied strains, three belonged to the Nostocales: Nostoc sp. (PMC 881.14), Aliinostoc sp. 48 49 (PMC 882.14) and Calothrix sp (PMC 884.14). 50 51 52 Nostoc sp. (Vaucher ex Bornet & Flahault, 1886) 53 54 Nostoc sp. (PMC 881.14) was isolated from the epilithic biofilm on the wall of the retention basin. Field 55 samples had a dense thallus with blackish green or brownish macroscopic colonies (Fig. 15). These 56 57 macroscopic colonies resulted from several small, amorphous, fine, thin and mucilaginous colonies. In 58 59 culture, old colonies (up to 130 µm in diameter) were enveloped by a firm periderm that could break and 60 release young colonies. Within the colonies, the filaments were very densely tangled, flexuous and/or

11 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. coiled. They were blackish green or brownish, ~5 µm wide, highly constricted at cross-walls, uniseriate, 1 2 unbranched, isopolar, moniliform and/or curled (Fig. 15). Cells were isodiametric, well separated from one 3 4 another by cross-walls, wider than long, 2.8-5.5 (4.5±0.9) μm wide, 2.6-4.6 (3.4±0.5) µm length with a 5 length:width ratio of 0.7. The apical cells were spherical to conical (data not shown). The heterocytes were 6 7 2.6-5.7 (3.4±0.6) µm wide and 2.3-5.1 (3.8±0.7) µm long. Akinetes were never observed during the three 8 9 years of culture, even when the strain was cultivated in Z8X medium without a nitrogen source to induce 10 11 stress conditions. An extensive gelatinous matrix was observed resulting in a long internal diffusion path 12 from the surface to the trichomes. A sheath with a periplasmic membrane was clearly visible by TEM (Fig. 13 14 15). The thylakoids were fascicular with irregular spherical formations (Fig. 15). 15 16 The 16S rRNA gene sequence analysis showed that PMC 881.14 strain shared ≥ 96% sequence identity 17 18 with other Nostoc sp. strains (data not shown). The phylogenetic tree based on 16S rRNA gene sequences 19 showed that the PMC 881.14 strain was grouped into a unique Nostoc cluster (Fig. 14) with a high 20 21 bootstrap value. Within this cluster three sequences were not assigned at the species level, and the five 22 23 others were assigned to different species: N. calcicola, N. punctiforme, N. flagelliforme, N. edaphicum, N. 24 ellisporum. 25 26 27 28 It is now well known that Nostoc is a complex genus. The species belonging to Nostoc were generally 29 30 described as microscopic, spherical, oval, ovoid, irregular in their colony form, gelatinous, irregularly 31 clustered, amorphous, dull olive to brownish gelatinous clusters free-living on mud (Table S8). Because of 32 33 this heterogeneity, it is difficult to clearly differentiate related taxa within the genus based solely on 34 35 morphology (Singh et al. 2016 and 2020). Previous studies using a polyphasic approach led to the splitting 36 37 of Nostoc into several genera, including Mojavia, Desmonostoc and Compactonostoc (Hrouzek et al. 2003; 38 Řeháková et al. 2007, Cai et al, 2019). It is difficult to separate these groups within Nostoc sensu lato based 39 40 only on morphological characteristics (Table S8). Following the concept that a taxonomic unit (family, 41 42 genus, species) must be included in only one phylogenetic lineage (Komárek, 2018), the strain PMC 43 881.14 was considered as a Nostoc sp. because our results did not allow us to give any further species 44 45 assignment to this strain. 46 47 48 49 Aliinostoc sp. (S.N. Bagchi, N. Dubey & P. Singh, 2017) 50 Aliinostoc sp. (PMC 882.14) was isolated from the epilithic biofilm on the wall of the retention basin. Field 51 52 samples showed free blue-green filaments (Fig. 16), while 53 54 in culture the filaments were flexuous and entangled. The trichomes were pale blue-green, cylindrical, thin 55 (< 6 µm wide), and highly constricted at cross-walls (Fig. 16). Cells were well separated from each other 56 57 by cross-walls, isodiametric, and longer than wide, 3.4-5.6 (4.5±0.6) μm wide by 2.6-6.4 (4.6±1.0) µm long 58 59 with a length:width ratio of 1.0 (Table S9). SEM showed that apical cells were conical (Fig. 16). The 60 heterocytes were 4.1-6.5 (5.4±0.5) µm wide by 4.1-6.8 (5.4±0.6) µm length and akinetes were 4.5-7.4

12 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. (5.9±0.8) µm wide by 4.5-9.0 (7.2±1.0) µm long (Table S9). No sheath was visible under TEM (Fig. 16). 1 2 The thylakoids were fascicular with irregular spherical formations (Fig. 16). The cells contained several 3 4 reserve granules and numerous carboxysomes (Fig. 16). The heterocyst TEM observations showed a few 5 thylakoids and large cyanophycin granules. 6 7 The 16S rRNA gene sequence analysis showed that PMC 882.14 shared ≥ 98% sequence identity with 8 9 other Aliinostoc sp. Strains (data not shown). The trees based on 16S rRNA gene sequences placed PMC 10 11 882.14 within a well-grouped cluster of Aliinostoc sequences (Fig. 14). Within this cluster, several 12 sequences were identified as Aliinostoc tiwarii, A. constricutum, A. soli, A. magnkinetifex, A. catenatum 13 14 and A. morphoplasticum, mainly based on morphological characteristics (Table S9). Aliinostoc sp. Showed 15 16 close morphological resemblance to the genera Nostoc and Trichormus. A diacritical feature for Aliinostoc 17 18 is the presence of motile hormogonia with gas vesicles (Bagchi et al. 2017). The authors also stated that 19 filaments must be loosely arranged with variable tendencies for coiling. These organisms were generally 20 21 isolated from habitats rich in dissolved ions and salts, with high water conductivity. As for the Nostoc sp. 22 23 PMC 881.14, description and morphology alone were of little help in assigning the strain to Aliinostoc 24 sensu lato (Bagchi et al. 2017) (Table S9). Based on the rule that a taxonomic unit (i.e. genus, species) 25 26 must be included in only one phylogenetic lineage (Komárek, 2018), the strain PMC 882.14 was named 27 28 Aliinostoc sp. because our data were not sufficient to give a species assignment. 29 30 31 Calothrix sp. (C. Agardh ex Bornet & Flahaut, 1886) 32 33 Calothrix sp. (PMC 884.14) was isolated from the biofilm at the surface of the mud of the retention basin. 34 35 Observations of field samples showed a brown filamentous thallus basally attached to the substratum and 36 37 forming thin mats. In culture, the filamentous thallus formed small brownish-green tufts (Fig. 17). 38 Filaments were aggregated, flexuous, heteropolar, widened near the base and narrowed towards the ends, 39 40 without false branching. The terminal cells were of variable length, conical, bluntly pointed and tapered. 41 42 The sheath was generally firm and colorless. The trichomes were of different lengths (> 800 µm long), pale 43 blue-green, olive-green to greyish-green, more or less constricted at the cross-walls, and narrowed at the 44 45 apex: 6.7-10.4 (8.1±0.7) µm wide at the base, 4.7-7.7 (5.8±0.6) µm in the middle and 3.5-5 (4.4±0.4) µm 46 47 near the ends (Fig. 17, Table S10). Vegetative cells were more or less quadratic, generally shorter than 48 49 wide in the upper part of the trichome and/or longer than wide in the lower part of the trichome, 5.2-7.9 50 (6.6±0.7) μm wide by 4.8-6.2 (5.5±0.4) µm long. Heterocytes were basal, single, hemispherical, 4.7-8.2 51 52 (6.2±0.9) μm wide by 3.5-7.3 (4.9±0.9) µm long. Akinetes were not observed during the three years the 53 54 cultures were maintained, even when strain PMC 884.14 was cultivated under stress conditions. 55 The 16S rRNA gene sequence analysis showed that PMC 884.14 shared ≥ 99% sequence identity with 56 57 other Calothrix sp. strains (data not shown). Phylogenetic trees grouped PMC 884.14 into a coherent 58 59 Calothrix clade (Fig. 14) in which the sequences were mainly assigned to Calothrix sp. Recent revision 60 suggested that the genus Calothrix should be split into several genera including Tolypothrix and Rivularia

13 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. (Berrendero et al. 2016). Based on a polyphasic study of 135 heterocytous tapering cyanobacteria, of which 1 2 99 were identified as Calothrix, these authors showed that the majority of Calothrix sequences were spread 3 4 among four lineages, two of which were marine, while the other two were freshwater and soil strains 5 (Berrendero et al. 2011, 2016). As the sequences for the Calothrix type species (C. confervicola) were 6 7 lacking, and no culture was available in any collection, we could not say which Calothrix clade represents 8 9 the true Calothrix. It would be helpful to conduct more phylogenetic analyses to elucidate the phylogeny of 10 11 the heteropolar, tapering taxa within Nostocales. Based on the rule that a taxonomic unit must be included 12 in only one phylogenetic lineage (Komárek, 2018), PMC 884.14 was assigned to Calothrix sp. as this study 13 14 did not allow us to give a species name to this strain. 15 16 17 18 Potential therapeutic properties of cyanobacterial strains from the muds of the thermal springs of 19 Balaruc-les-Bains 20 21 A polyphasic approach is still the best way to correctly determine the taxonomy of a new isolate (Komárek 22 23 2018, 2020). Using morphological, ultrastructural and molecular methods, the nine cyanobacterial isolates 24 from the muds of Thermes de Balaruc-Les-Bains, were clearly identified as belonging to the orders 25 26 Chroococcales: Pseudo-chroococcus couteii; Synechococcales: Leptolyngbya boryana; Oscillatoriales: 27 28 Planktothricoides raciborskii, Laspinema sp., Microcoleus vaginatus, Lyngbya martensiana and 29 30 Nostocales: Nostoc sp., Aliinostoc sp., Calothrix sp. This taxonomic diversity along with literature reports 31 of the bioactive metabolite synthesis potential of these taxa allowed us to hypothesize that some of the 32 33 metabolites produced by these strains may be active in the mud therapy at Thermes de Balaruc-Les-Bains. 34 35 A recent literature review (Demay et al. 2019) emphasized this great potential (Table S11). All 36 37 cyanobacteria are known to synthesize pigments such as chlorophylls, phycobiliproteins and carotenoids 38 (Fig. S1), which can have antioxidant or anti-inflammatory properties (Table S11). These pigments were 39 40 synthesized by the studied isolates in different percentages, depending on the strain and the physiology 41 42 (Table S11). In addition to pigment production, Lyngbya, Calothrix and Chroococcus strains have attracted 43 further interest because they produce bioactive molecules such as mycosporine-like amino acids (MAAs) 44 45 and the yellow-brown scytonemin pigments with reported antioxidant, anti-inflammatory and UV- 46 47 protecting activities. Numerous bioactive compounds from strains belonging to the Nostoc genus have also 48 49 been described, including the anti-inflammatory metabolites aeruginosins and scytonemins. 50 Planktothricoides, Aliinostoc, Laspinema sp., Microcoleus and the new genus Pseudo-chroococcus were 51 52 not reported in Demay et al. (2019) to have antioxidant and/or anti-inflammatory activities. 53 54 Misidentification of Oscillatoriales with their very simple filamentous organization and no diacritical 55 characteristics, occurs frequently and some isolates could have been described as Phormidium, Lyngbya or 56 57 other simple organized Oscillatoriales. 58 59 60 CONCLUSIONS

14 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Cyanobacterial taxonomy has changed substantially over the last few years thanks to revisions of the 1 2 morphological criteria and the use of gene sequencing. To resolve the taxonomic discrepancies between 3 4 morphological and 16S rRNA data and better assign new isolates of cyanobacteria, the use of whole 5 genome sequencing is warranted. Approximately 1440 cyanobacterial genomes (complete or in progress) 6 7 are currently available (S. Halary, personal communication) in GenBank (NCBI Datasets “cyanobacteria”, 8 9 accessed on 9 Sept. 2020). However, this growing number of available genomes may hide the low 10 11 representativeness of the diversity of cyanobacteria since most sequenced strains belong to the orders 12 Nostocales and Synechococcales with 457 and 600 available assemblies, respectively. For example, very 13 14 few genomes were available for the genera assigned to Balaruc strains with 2-8 genomes available for 15 16 Planktothricoides, Lyngbya and Microcoleus and none for Aliinostoc and Pseudo-chroococcus. Correct 17 18 assignment of new isolates is essential for understanding the relationships among strains and identifying 19 beneficial therapeutic properties such as those possessed by the mud therapy strains from Thermes de 20 21 Balaruc-Les-Bains. The isolates maintained in collections will allow in-depth investigation of their 22 23 potential for production of bioactive metabolites using genomic and biochemical methods. 24 25 26 CONFLICT OF INTEREST 27 28 All authors declare that there is no conflict of interest regarding the publication of this paper. 29 30 31 AUTHOR CONTRIBUTIONS 32 33 SH, CY, CD and CB have worked on the design of the experiments. CD, SH, BP, GT and JD have 34 35 performed the experiments. CD, SH, BP and CB have performed the data analysis. CD, SH, JD, SD, BM, 36 37 CY and CB have written and edited the paper. 38 39 40 ACKNOLEDMENTS 41 42 This work was supported by the ANRT through a PhD grant awarded to J. Demay. We would like to thank 43 the UMR 7245 MCAM, Muséum National d’Histoire Naturelle, Paris, France for laboratories facilities and 44 45 the Thermes de Balaruc-Les-Bains for complimentary founds. The authors thanks Dr P. Méric (Thermes of 46 47 Balaruc-Les-Bains) for his support during prospecting campaigns on and C. Djediat, A. Marme and S. 48 49 Jaber for preliminary TEM studies. This work was supported by the “Société Publique Locale 50 d’Exploitation des Thermes de Balaruc-Les-Bains” (SPLETH), the town of Balaruc-Les-Bains and the 51 52 National Muséum of Natural History. The authors also would like to thank Peerwith 53 54 (https://www.peerwith.com) for English language editing. 55 56 57 DEDICATION 58 59 This manuscript is dedicated to the memory of late Professor Alain Couté, who made an outstanding 60 contribution to the taxonomy of cyanobacteria and microalgae in the last decades. His work covers a wide

15 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. range of studies dealing with photosynthetic microorganisms from marine and freshwater habitats, and also 1 2 from terrestrial and extreme environments. Alain Couté’s contribution will be always relevant and was a 3 4 driver for shaping this manuscript. 5 6 7 REFERENCES 8 9 Bagchi, S. N., Dubey, N., and Singh, P. (2017). Phylogenetically distant clade of Nostoc-like taxa with the 10 11 description of Aliinostoc gen. nov. and Aliinostoc morphoplasticum sp. nov. Int. J. Syst. Evol. Microbiol. 12 67, 3329–3338. doi:10.1099/ijsem.0.002112. 13 14 Baudinat, C. (1986). Contribution à l’étude de la maturation de péloïdes : application aux stations thermales 15 16 de Balaruc-les-bains (34) et Cransac (12). 17 18 Bernard, C., Ballot, A., Thomazeau, S., Maloufi, S., Furey, A., Mankiewicz-Boczek, J., et al. (2017). 19 “Cyanobacteria Associated with the production of ,” in Handbook of Cyanobacterial 20 21 Monitoring and Analysis, 501. 22 23 Berrendero Gómez, E., Johansen, J. R., Kaštovský, J., Bohunická, M., and Čapková, K. (2016). 24 Macrochaete gen. nov. (Nostocales, Cyanobacteria), a taxon morphologically and molecularly distinct 25 26 from Calothrix. J. Phycol. 52, 638–655. doi:10.1111/jpy.12425. 27 28 Berrendero, E., Perona, E., and Mateo, P. (2011). Phenotypic variability and phylogenetic relationships of 29 30 the genera Tolypothrix and Calothrix (Nostocales, Cyanobacteria) from running water. Int. J. Syst. Evol. 31 Microbiol. 61, 3039–3051. doi:10.1099/ijs.0.027581-0. 32 33 Cai, F., Li, X., Geng, R., Peng, X., and Li, R. (2019). Phylogenetically distant clade of Nostoc-like taxa with 34 35 the description of Minunostoc gen. nov. and Minunostoc cylindricum sp. nov. Fottea 19, 13–24. 36 37 doi:10.5507/fot.2018.013. 38 Cellamare, M., Duval, C., Drelin, Y., Djediat, C., Touibi, N., Agogué, H., et al. (2018). Characterization of 39 40 phototrophic microorganisms and description of new cyanobacteria isolated from the saline-alkaline crater- 41 42 lake Dziani Dzaha (Mayotte, Indian ). FEMS Microbiol. Ecol. 94. doi:10.1093/femsec/fiy108. 43 Cuzman, O. A., Ventura, S., Sili, C., Mascalchi, C., Turchetti, T., D’Acqui, L. P., et al. (2010). Biodiversity 44 45 of Phototrophic Biofilms Dwelling on Monumental Fountains. Microb. Ecol. 60, 81–95. 46 47 doi:10.1007/s00248-010-9672-z. 48 49 Demay, J., Bernard, C., Reinhardt, A., and Marie, B. (2019). Natural Products from Cyanobacteria: Focus on 50 Beneficial Activities. Mar. Drugs 17, 320. doi:10.3390/md17060320. 51 52 Demay, J., Halary, S., Knittel-Obrecht, A., Villa, P., Duval, C., Hamlaoui, S., et al. (2020). Antioxidant and 53 54 anti-inflammatory properties of cyanobacteria from thermal mud (Balaruc-Les-Bains, France): a multi- 55 approach study. In prep. 56 57 Dupuis, E. (1987). Pré-étude relative à l’évaluation de la production algale des eaux thermales de Balaruc- 58 59 les-Bains. 60

16 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Gama, W. A., Rigonato, J., Fiore, M. F., and Sant’Anna, C. L. (2019). New insights into Chroococcus 1 2 (Cyanobacteria) and two related genera Cryptococcum gen. nov. and Inacoccus gen. nov. Eur. J. Phycol. 3 4 54, 315–325. doi:10.1080/09670262.2018.1563913. 5 Grossman, A. R. (2003). A molecular understanding of complementary chromatic adaptation. Photosynth. 6 7 Res. 76, 207–215. doi:10.1023/A:1024907330878. 8 9 Guidi-Rontani, C., Jean, M. R. N., Gonzalez-Rizzo, S., Bolte-Kluge, S., and Gros, O. (2014). Description of 10 11 new filamentous toxic Cyanobacteria (Oscillatoriales) colonizing the sulfidic periphyton mat in marine 12 mangroves. FEMS Microbiol. Lett. 359, 173–181. doi:10.1111/1574-6968.12551. 13 14 Hamlaoui, S. (2014). Isolement, culture et analyses toxicologiques de souches de micro-algues et de 15 16 cyanobactéries des eaux thermales de Balaruc-les-Bains. 17 18 Hrouzek, P., Simek, M., and Komárek, J. (2003). Nitrogenase activity (acetylene reduction activity) and 19 diversity of six soil Nostoc strains. Arch. Hydrobiol. Suppl.146, 87–101. 20 21 Katoh, K., Rozewicki, J., and Yamada, K. D. (2019). MAFFT online service: multiple sequence alignment, 22 23 interactive sequence choice and visualization. Brief. Bioinforma. 20, 1160–1166. doi:10.1093/bib/bbx108. 24 Komárek J., J. J. R. (2015). Filamentous Cyanobacteria. In: Wehr J.D., Sheath R.G & Kociolek J. P. [Eds]: 25 26 Freshwater Algae of North America. Ecology and Classification. Academic Press, New York, USA, pp. 27 28 135-235. 29 30 Komárek, J., and Anagnostidis, K. (2005). Cyanoprokaryota 2.Teil: Oscillatoriales. Elsevier GmbH, 31 Munchen, 759 p. 32 33 Komárek, J. (2020). Quo vadis, taxonomy of cyanobacteria (2019). Fottea 20, 104–110. 34 35 doi:10.5507/fot.2019.020. 36 37 Komárek, J. (2007). Phenotype diversity of the cyanobacterial genus Leptolyngbya in the maritime 38 Antarctic. Pol. Polar Res. 28 (3): 211–231. 39 40 Komárek, J. (2010). Recent changes (2008) in cyanobacteria taxonomy based on a combination of molecular 41 42 background with phenotype and ecological consequences (genus and species concept). Hydrobiologia 639, 43 245–259. doi:10.1007/s10750-009-0031-3. 44 45 Komárek, J. (2017). Several problems of the polyphasic approach in the modern cyanobacterial system. 46 47 Hydrobiologia 811. doi:10.1007/s10750-017-3379-9. 48 49 Komárek, J. (2018). Delimitation of the family (Cyanobacteria) according to the modern 50 polyphasic approach (introductory review). Brazilian J. Bot. 41, 449–456. doi:10.1007/s40415-017-0415-y. 51 52 Komárek, J., and Anagnostidis, K. (2007). Süßwasserflora von Mitteleuropa, Bd. 19/2: Cyanoprokaryota: 53 54 Bd. 2 / Part 2: Oscillatoriales. Springer Spektrum Available at: 55 https://www.springer.com/gp/book/9783827419149 [Accessed July 8, 2020]. 56 57 Komárek, J., Kaštovský, J., Mareš, J., and Johansen, J. R. (2014). Taxonomic classification of 58 59 cyanoprokaryotes (cyanobacterial genera) 2014, using a polyphasic approach. Preslia 86, 295-335. 60

17 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Komárek, J., and Komárková, J. (2004). Taxonomic review of the cyanoprokaryotic genera Planktothrix and 1 2 Planktothricoides. Czech Phycology 4:1, 1-18. 3 4 Komárková, J., Jezberová, J., Komárek, O., and Zapomělová, E. (2010). Variability of Chroococcus 5 (Cyanobacteria) morphospecies with regard to phylogenetic relationships. Hydrobiologia 639, 69–83. 6 7 doi:10.1007/s10750-009-0015-3. 8 9 Kumar, S., Stecher, G., and Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis 10 11 Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 33, 1870–1874. doi:10.1093/molbev/msw054. 12 Lokmer, A. (2007). Polyphasic approach to the taxonomy of the selected oscillatorian strains 13 14 (Cyanobacteria). Master thesis, University of South Bohemia, Faculty of Biological Sciences, Cezke 15 16 Budejovice 40 pp. 17 18 Lowe, T. M., and Chan, P. P. (2016). tRNAscan-SE On-line: integrating search and context for analysis of 19 transfer RNA genes. Nucleic Acids Res. 44, W54--57. doi:10.1093/nar/gkw413. 20 21 Parveen, B., Ravet, V., Djediat, C., Mary, I., Quiblier, C., Debroas, D., et al. (2013). Bacterial communities 22 23 associated with Microcystis colonies differ from free-living communities living in the same ecosystem: 24 Bacterial diversity inside Microcystis colonies. Environ. Microbiol. Rep., n/a--n/a. doi:10.1111/1758- 25 26 2229.12071. 27 28 Rajaniemi, P., Hrouzek, P., Kastovská, K., Willame, R., Rantala, A., Hoffmann, L., et al. (2005). 29 30 Phylogenetic and morphological evaluation of the genera Anabaena, Aphanizomenon, Trichormus and 31 Nostoc (Nostocales, Cyanobacteria). Int. J. Syst. Evol. Microbiol. 55, 11–26. doi:10.1099/ijs.0.63276-0. 32 33 Řeháková, K., Johansen, J. R., Casamatta, D. A., Xuesong, L., and Vincent, J. (2007). Morphological and 34 35 molecular characterization of selected desert soil cyanobacteria: three species new to science including 36 37 Mojavia pulchra gen. et sp. Nov. Phycologia 46, 481–502. doi:10.2216/06-92.1. 38 Rippka, R. (1988). Isolation and purification of cyanobacteria. Meth. Enzym. 167, 3–27. 39 40 Roldán, M., Ramírez, M., Del Campo, J., Hernández-Mariné, M., and Komárek, J. (2013). Chalicogloea 41 42 cavernicola gen. nov., sp. nov. (Chroococcales, Cyanobacteria), from low-light aerophytic environments: 43 combined molecular, phenotypic and ecological criteria. Int. J. Syst. Evol. Microbiol. 63, 2326–2333. 44 45 doi:10.1099/ijs.0.045468-0. 46 47 Singh, P., Shaikh, Z. M., Gaysina, L. A., Suradkar, A., and Samanta, U. (2016). New species of Nostoc 48 49 (cyanobacteria) isolated from Pune, India, using morphological, ecological and molecular attributes. 50 Syst Evol 302, 1381–1394. doi:10.1007/s00606-016-1337-z. 51 52 Singh, P., Šnokhousová, J., Saraf, A., Suradkar, A., and Elster, J. (2020). Phylogenetic evaluation of the 53 54 genus Nostoc and description of Nostoc neudorfense sp. nov., from the Czech Republic. Int. J. Syst. Evol. 55 Microbiol. 70, 2740–2749. doi:10.1099/ijsem.0.004102. 56 57 Strunecký, O., Komárek, J., Johansen, J., Lukešová, A., and Elster, J. (2013). Molecular and morphological 58 59 criteria for revision of the genus Microcoleus (Oscillatoriales, Cyanobacteria). J. Phycol. 49, 1167–1180. 60 doi:10.1111/jpy.12128.

18 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Suda, S. (2002). Taxonomic revision of water-bloom-forming species of oscillatorioid cyanobacteria. Int. J. 1 2 Syst. Evol. Microbiol. 52, 1577–1595. doi:10.1099/ijs.0.01834-0. 3 4 Wood, S. A., Rhodes, L., Smith, K., Lengline, F., Ponikla, K., and Pochon, X. (2017). Phylogenetic 5 characterisation of marine Chroococcus -like (Cyanobacteria) strains from the Pacific region. New Zeal. J. 6 7 Bot. 55, 5–13. doi:10.1080/0028825X.2016.1205634. 8 9 Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids 10 11 Res. 31, 3406–3415. doi:10.1093/nar/gkg595. 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

19 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 FIGURES LEGENDS 4 5 6 7 Figure 1. Retention basin of the Thermes de Balaruc-Les-Bains. Views of the mud 8 9 maturation basin (mb), the water retention basin (rb) and the thermal spring water supply (ts) 10 on February 28th, 2014 (A) and on May 12th, 2014 (B). 11 12 13 14 Figure 2. Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences of 15 16 representative cyanobacteria strains (129 sequences, 1446 aligned nucleotide positions, 17 GTR+G+I model) belonging to the orders Oscillatoriales, Nostocales, Chroococcales, 18 19 Synechococcales and the studied strains form Thermes de Balaruc-Les-Bains (in bold). 20 21 Gloeobacter violaceus was used as outgroup. Numbers above branches indicate bootstrap 22 support (>50% shown) based on 1000 replicates. 23 24 25 26 Figure 3. Light microscopy (A), SEM (B) and TEM (C-F) micrographs of Pseudo- 27 28 chroococcus couteii (PMC 885.14) from Thermes de Balaruc-Les-Bains. A: Light microscopy 29 of a packet-form colony, four-celled groups of spherical, very small cells, brownish color with 30 31 dense mucilaginous sheath around cells. B: external observation of a packet-form colonies C, 32 33 D, E, F.: the four-grouped cells were surrounded by a dense mucilage. Cells details showing a 34 35 fasciculate arrangement of thylakoids. Abbreviations: c: colony of four-grouped cells, cy: 36 cyanophycine granules, cw: cell wall, pa: cells partition, mu: mucilage, t: thylakoids. 37 38 39 40 Figure 4. Consensus phylogenetic tree (Maximum likelihood tree presented) based on 16S 41 rRNA-encoding gene sequences (54 sequences, 705 aligned nucleotide positions, GTR+G+I 42 43 model) of representative cyanobacteria strains belonging to the order Chroococcales. Thermes 44 45 de Balaruc-Les-Bains’s strain is indicated in bold, other species and sequences were obtained 46 47 from Genbank. Gloeobacter violaceus PCC 7421 was used as an out-group. Numbers above 48 49 branches indicate bootstrap support (>50%) from 1,000 replicates. Bootstrap values are given 50 in the following order: maximum likelihood / neighbor joining / maximum parsimony. (✷ 51 52 marine strains; Cluster 1 : Chroococcus-like , 2 : Limnococcus/Gloeocapsa, 3 : 53 54 Inacoccus/Cryptococcum, 4 : Chroococcus sensu stricto) 55 56 57 Figure 5. Consensus phylogenetic tree (Maximum likelihood tree presented) based on 16S- 58 59 23S ITS gene sequences (21 sequences, 1915 aligned nucleotide positions, GTR+G+I model) 60

cw bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 of representative cyanobacteria strains belonging to the orders Chroococcales, Thermes de 4 5 Balaruc-Les-Bains’s strains (in bold), other species were obtained from Genbank. Microcystis 6 7 aeruginosa PMC 728.11 was used as an outgroup. Numbers above branches indicate 8 bootstrap support (>50%) from 1000 replicates. Bootstrap values are given in the following 9 10 order: maximum likelihood / neighbor joining / maximum parsimony. 11 12 13 14 Figure 6. 16S-23S ITS secondary structure of the D1-D1’ helices of Inacoccus, 15 Cryptococcum, Chroococcus and Chroococcus-like (syn. Pseudo-chroococcus) cluster. 16 17 18 19 Figure 7. Light microscopy (A, B), MEB (C) and TEM (D-F) micrographs of Leptolyngbya 20 boryana (PMC 883.14) from Thermes de Balaruc-Les-Bains. A: entangled filaments and 21 22 trichomes with sheath. B: observations of trichomes with probable exudates localized on cell 23 24 junctions. Longitudinal (C-E) and cross sections (F) of trichomes with sheaths showing the 25 26 ultrastructural details. Abbreviations: cc: cross-wall constriction, cm: cytoplasmic membrane, 27 cy: cyanophycin granules, t: thylakoids, tz: transparent zone. 28 29 30 31 Figure 8. Consensus phylogenetic tree (Maximum likelihood tree presented) based on 16S 32 33 rRNA gene sequences (54 sequences, 1333 aligned nucleotide position, GTR+G+I model) of 34 representative cyanobacteria strains belonging to the orders Synechococcales, Thermes de 35 36 Balaruc-Les-Bains’s strain is indicated in bold (with brown color), other species were 37 38 obtained from Genbank. Gloeobacter violaceus PCC 7421 was used as an outgroup. Numbers 39 above branches indicate bootstrap support (>50%) from 1000 replicates. Bootstrap values are 40 41 given in the following order: maximum likelihood / neighbor joining / maximum parsimony. 42 43 44 45 Figure 9. Consensus phylogenetic tree (Maximum likelihood tree presented) based on 16S 46 rRNA gene sequences (57 sequences, 1305 aligned nucleotide positions, GTR+G+I model) of 47 48 representative cyanobacteria strains belonging to the orders Oscillatoriales, Thermes de 49 50 Balaruc-Les-Bains’s strains are indicated in bold (colored in blue), other species were 51 obtained from Genbank. Gloeobacter violaceus PCC 7421 was used as an out-group. 52 53 Numbers above branches indicate bootstrap support (>50%) from 1000 replicates. Bootstrap 54 55 values are given in the following order: maximum likelihood / neighbor joining / maximum 56 57 parsimony. 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Figure 10. Light microscopy (A, B), SEM (C) and TEM (D-F) micrographs of 4 5 Planktothricoides raciborskii (PMC 877.14) from Thermes de Balaruc-Les-Bains. A: Light 6 7 microscopy of trichomes from field sample. B: Light microscopy of trichomes in culture. C: 8 trichomes associated or not with bacteria. Longitudinal (C-E) and cross sections (F) of the 9 10 trichomes showing the ultrastructural details. Abbreviations: ac: apical cell, ae: aerotopes, 11 12 bact: bacteria, c: carboxysome, cm: cytoplasmic membrane, cy: cyanophycin granules, its: 13 14 inter thylakoidal space, r: reserves (lipids, polyphosphates, poly-β-hydroxybutyrate), t: 15 thylakoids, vs: clear vesicle. 16 17 18 19 Figure 11. Light microscopy (A), SEM (B) and TEM (C-D-E-F) micrographs of Laspinema 20 sp. (PMC 878.14) from Thermes de Balaruc-Les-Bains. A: filaments with typical shape of 21 22 apical cell. B: observations of filaments. Longitudinal (C-E) and cross sections (F) of 23 24 trichomes showing the ultrastructural details. Abbreviations: ab: beta-hydroxybutyric acid, ac: 25 26 apical cell, ca: carboxysome, cy: cyanophycin granule, gv: gas vesicles, its: inter thylakoidal 27 space, t: thylakoids. 28 29 30 31 Figure 12. Light microscopy (A), SEM (B) and TEM (C-D-E-F) micrographs of Microcoleus 32 33 vaginatus (PMC 879.14) from Thermes de Balaruc-Les-Bains. A: entangled filaments with 34 sheath. B: filaments with apical cell. Longitudinal (C-D-E-F) of trichomes with sheaths 35 36 showing the ultrastructural details. Abbreviations: ab: acid beta-hydroxybutyric, ac: apical 37 38 cell, cy: cyanophycin granule, f: filament, t: thylakoids. 39 40 41 Figure 13. Light microscopy (A), SEM (B) and TEM (C-D-E-F) micrographs of Lyngbya 42 43 martensiana (PMC 880.14) from Thermes de Balaruc-Les-Bains. A: filaments with sheath. B: 44 45 observation of a filament. Longitudinal section (C-D-F) and cross section showing the 46 ultrastructural details. Abbreviations: ab: beta-hydroxybutyric acid, ca: carboxysome, cy: 47 48 cyanophycin, cw: cell wall, its: inter thylakoidal space, f: filament, pbs: phycobilisomes 49 50 (photosynthetic antenna), s: sheath, t: thylakoids. 51 52 53 Figure 14. Consensus phylogenetic tree (Maximum likelihood tree presented) based on 16S 54 55 rRNA gene sequences (69 sequences, 1310 aligned nucleotide position, GTR+G+I model) of 56 57 representative cyanobacteria sequences (Genbank) belonging to the Nostocales and the 58 studied strains from Thermes de Balaruc-Les-Bains are indicated in bold (with purple color). 59 60 Gloeobacter violaceus PCC 7421 was used as an out-group. Numbers above branches bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 indicate bootstrap support (>50%) from 1000 replicates. Bootstrap values are given in the 4 5 following order: maximum likelihood / neighbor joining / maximum parsimony. 6 7 8 Figure 15. Light microscopy (A-B), SEM (C) and TEM (D-E-F-G) micrographs of Nostoc sp. 9 10 (PMC 881.14) from Thermes de Balaruc-Les-Bains. A: Observation of the filaments located 11 12 inside the capsule, B: Observation of two colonies, spherical, irregular in their shape, dull 13 14 olive to brownish-colored and gelatinous. Filaments very densely entangled, flexuous, coiled 15 inside the colonies. C: external observation of a capsule. Longitudinal (D-F-G) and cross 16 17 sections (E) showing the ultrastructural details. Abbreviations: cp: capsule, cw: cell wall, f: 18 19 filament, mu: mucilage, sh: sheath, t: thylakoids. 20 21 22 Figure 16. Light microscopy (A), SEM (B) and TEM (C-F) micrographs of Aliinostoc sp. 23 24 (PMC 882.14) from Thermes de Balaruc-Les-Bains. A: Light microscopy of filament from 25 26 field sample. B: SEM observations of trichome. Longitudinal sections(C-F) of an heterocyte 27 (E) and vegetative cells (C, D, F) showing the ultrastructural details. Abbreviations: ca: 28 29 carboxysome, cif: carboxysome in formation, cw: cell wall, its: inter thylakoidal space, t: 30 31 thylakoids. 32 33 34 Figure 17. Light microscopy (A), SEM (B) and TEM (C-D-E-F) micrographs of Calothrix sp. 35 36 (PMC 884.14) from Thermes de Balaruc-Les-Bains. A: entangled filaments with sheath. B: 37 38 observations of filament. Longitudinal (C-D-F) and cross sections (E) of trichomes with 39 sheaths showing the ultrastructural details. Abbreviations: ca: carboxysome, ld: lipid droplet, 40 41 cw: cell wall, sh: sheath, t: thylakoids, mu: mucilage. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 Fig. 1 2 3 4 5 6 A B 7 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this 8 preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in 9 perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 10 11 12 rb 13 14 15 ts 16 17 rb mb 18 mb 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Phormidium

Gloeobacter violaceus

Laspinema thermale Laspinema etoshii 1 Laspinema thermale Fig. 2 cf. Laspinema terebriformis (NZ CP0247851:81819-83309) 2 Laspinema lumbricale 3 PlanktothricoidesPlanktothricoidesPlanktothricoides raciborskii raciborskii raciborskii

sp. PMC 878.14 (MT984288) CCNUN1

4 PCC 73102 KR2008/49 (KC014068.1) Planktothricoides raciborskii HK S3 clone cl2 (MF360982.1)PCC 7421 (AF132790.1) I (AJ630451.1) KR2003/25 (AY575936.1) V HK S5 clone cl2 (MF360989.1) X 5 III Lukesova 1/87 (AM711523.1) (NZ AP0182221:c3653887-3652392) 6 UTCC 476 (AF218375.1) Lyngbya robusta ATCC 29413 (HF678501.1) sp. PCC 7422 (HG004586.1) 7 sp. PCC 9709 (AF027654.1) NIES-207 (AB045960.1) sp. ATCC 53789 (AF062638.1) sp. PCC 9230 (HG004585.1) PMC 877.14OR1-1 (MT984287) (B045964.1) sp. PMC 881.14 (MT984289) NIES-25 Microcoleus vaginatus 1 8 MicrocoleusMicrocoleus vaginatus vaginatus Lyngbya aestuarii Nostoc flagelliforme

Nostoc punctiforme NSLA4 (AB045963.1) Nostoc Nostoc calcicola Nostoc Nostoc edaphicum 9 CCALA 966 (JN854138.1) Nostoc Kom-BAI/1983 Nostoc ellipsosporum Microcoleus vaginatus Nostoc muscorum Desmonostoc muscorum sp. PCC 7120

74 Desmonostoc sp. PCC 7524 51 Desmonostoc CHAB 5844-1 (MK045295.1) 10 90 Nostoc linckia Microcoleus vaginatus 69 81 Trichormus variabilis 100 sp. PCC 9426 (AM711538.1) JR6 (JN230339.1)PCC 7419 Nostoc CHAB 5843 clone 3 (MK045297.1) Microcoleus vaginatus PMC 879.14 (MT984285) 98 11 JR12 (JN230335.1) 100 Nostoc Trichormus doliolum sp. PCC 6720 (DQ185240.1) 81 53 98 SAG 2502 (MK953007.1) 52 100 Nostoc 94 sp PCC 7107 Microcoleus autumnalis 86 Trichormus azollae 12 For100 Peer100 Review50 SA30 (MK503793.1) PCC 9802 (AF284803.1) Nostoc 99 CCALA 154 (JN230342.1) Nostoc tiwarii LI PS (MH497064.1) 13 Microcoleus autumnalis 100 Minunostoc cylindricum 100 constrictum 78 Minunostoc cylindricum SAG 35.90 (EF654081.1) sp. PMC 882.14 (MT984291) 14 Microcoleus vaginatus 81 76 Aliinostoc ZH1(3) PS (MH497065.1)SA18 (MK503791.1) Arct-Ph5 (DQ493873.2) 55 100 Aliinostoc Kamptonema animale 83 Aliinostoc SA24 (MK503792.1) 15 Luznice (KC633959.1) 54 53 Aliinostoc soli NOS (KY403996.1) 84 Kamptonema animaleCCALA 771 (KP221932.1) 65 99 Aliinostoc magnkinetifex 16 Trichodesmium erythraeum 100 59 Aliinostoc catenatum INDIA92 (AM230700.1) Aliinostoc morphoplasticum sp. CENA548 (KX458492.1) IMS101 77 99 Aliinostoc 17 (113473942:3137158-3138645) 69 97 sp. 90 Trichodesmium hildebrandtii 100 62 Anabaena 08 10 18 (AF091322.1) 96 Anabaena tenericaulis PMC 196.03 100 99 82 Dolichospermum solitarium Trichodesmium thiebautii (AF091321.1) 100 65 94 Dolichospermum flos-aquae PMC 207.03 19 100 Lyngbya martensiana PMC 880.14 (MT984286) Cuspidothrix issatschenkoi 100 Trichormus variabilis 0tu37s7 20 H3b/7 (JN854142.1) 61 Lyngbya martensiana 100 Trichormus variabilis HINDAK 2001/4 (AJ630456.1) CCALA 759 (EU196638.1) Anabaena cylindrica 21 cf. irriguum 99 GREIFSWALD (AJ630457.1) 100 63 Anabaena bergii Phormidium CCALA 147 (AM398779.1) Nodularia PCC 7122 cf. nigrum 99 22 100 90 Nodularia spumigenaPMC 314.07 Phormidium CCIBt3506 (MF072351.1) 83 sp. PCC 73104 85 Calothrix 100 23 CCIBt3549 (MF072353.1) 100 Calothrix 100 Calothrix sp. PCC 6303 Chroococcus subviolaceus CCALA 702 (GQ375044.1) 100 Calothrix sp. PCC 8909 (AM230693.1)CCY9414 98 100 100 91 24 westii 67 Calothrix thermalis CCIBt3508 (MF072352.1) 99 sp. XP11C (AM230698.1) Chroococcus subviolaceus cf. 65 91 Calothrix desertica 62 sp. PMC 884.14 (MT984290) 25 100 Calothrix CCALA 055 (GQ375047.1) Calothrix parietina Chroococcus 99 SAG 41.79 (KM019988.1) 100 Leptolyngbya boryanum 86 Leptolyngbya boryana 26 95 95 Chroococcus turgidus 100 Leptolyngbya boryana sp. PCC 7103 (AM230700.1) CCIBt3410 (MF072345.1) 100 PCC 7715 (AM230701.1) 100 62 Leptolyngbya boryana 100 100 100 100 Leptolyngbya boryana 92 50 PCC 7102 (NR 114995.1) 27 Chroococcus minutus CCALA 054 (MF072346.1) Leptolyngbya crispata Svet06 (GQ375048.1) 87 96 100 Leptolyngbya 87 100 Leptolyngbya Chroococcus minutus 95 Leptolyngbya antarctica 28 100 Leptolyngbya antarctica CCAP 1410/10 (HF678479.1) Stenomitos

Stenomitos Nodosilinea Stenomitos rutilans

Leptolyngbya frigida

Nodosilinea Nodosilinea Leptolyngbya nostocorum

Leptolyngbya

Nodosilinea redekei redekei Limnothrix Leptolyngbya mycoidea Leptolyngbya valderiana 29 Limnothrix redekei Cryptococcum brasiliense CCIBt3418 (MF072348.1) (X84810.1) sp. PCC 73106 (AB039000.1) PCC 6306 (EF429290.1) CCIBt3475 (MF072349.1) 30 sp. SEV5-3-C2 (AY239592.1)IAM M-101 (AB245143.1) Limnococcus sp. clone limneticus Alla11otu7-1 16PMC (KP676759.1) 885.14 (MT985488) sp. SEV5-3-C28 (AY239595.1) Cryptococcum komarkovaum sp. PMC 304.07 (GQ859652.1) NIES-2135 (LC215287.1)

sp. Ru-0-2 (MH688849.1)

31 (KF246490.1) CENA523 sp. sp. PMC 302.07 (GQ859653.1 302.07 PMC sp.

Gloeocapsa (KF307 2910 UTEX nodulosa SEV4-3-C6PMC (AY239598.1) 883.14 (MT984284) sp. CENA322 (KT731143.1) CENA322 sp.

sp. SK40 (AB067576.1) sp. Zehnder 1965/U140 (HM 32 Limnococcus sp. PCC 8801 (AF296873.1)

Inacoccus carmineus HA7619-LM2 (KF417430 ANT.L67.1 (AY493572.1)

sp. KO11DG (AB067577.1) M 7.6(Q56) (GQ8596 272.06 PMC ANT.LWA.1 (AY493604.1) 33 165c (AJ505943.1) ANT.L53B.2 (AY49357 Inacoccus carmineus sp. WH 8902 (AY620238.1) Uncultured sp. KO30D1 (AB067579.1) 34 Gloeothece EE018(Q942.1) LEGE 06108 (HQ sp. PCC 6803 (AY224195.1) AC281( 7918.1) SABC022801 (K Cyanothece 3.1) UAM 387 (JQ07 sp. ATCC 51142 (AF132771.1) sp. PCC 6702 (AB041936.1) 35 Gloeothece Pseudo-chroococcus couteii Nostocales Cyanothece 36 Gloeocapsa Synechococcales 37 Chroococcales Cyanothece

38 Synechocystis Oscillatoriales Cyanobacteria sp. YW-2019b strain TP201716.1 clone 03 (MN215477.1) 1) 39 692.1) 40 Cyanobacteria sp. YW-2019b strain TP201716.4 clone 06 (MN215485.1) 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Fig. 3 4 5 6 7 8 A B 9 10 mu mu 11 12 13 14 15 16 17 c 18 19 20 21 20 µm 2 µm 22 four-grouped cells 2µm 23 24 C D pa 25 cw 26 27 t 28 29 30 31 32 33 34 mu 35 cy 36 2µm mu 4µm2µm 37 38 39 EE F 40 41 cy 42 mu cw 43 t 44 45 t 46 47 cy t 48 49 50 4µm mu 51 1µm 52 t 1µm 1µm 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Fig. 4 2 3 4 5 6 7 Cyanobacteria sp. YW-2019b strain TP201716.5 clone 01 (MN215486.1) 8 100 / 100 / 100 Cyanobacteria sp. YW-2019b strain TP201716.4 clone 06 (MN215485.1) 100 / 100 / 100 Cyanobacteria sp. YW-2019b strain TP201716.3 clone 05 (MN215481.1) 9 Cyanobacteria sp. YW-2019b strain TP201716.1 clone 03 (MN215477.1) 10 63 / 73 / - Pseudo-chroococcus couteii PMC 885.14 (MT985488) 11 100 / 100 / 99 Chroococcus-like CAWBG110 (KU513782.1) ✷ 57 / 52 / - Chroococcus-like CAWBG113 (KU513783.1) ✷ 12 87 / 87 / 79 Chroococcus-like CAWBG101 (KU513781.1) ✷ 1 13 Gloeocapsa sp. PCC 73106 (AB039000.1) 14 98 / 98 / 98 Uncultured Limnococcus sp. clone Alla11otu7-1 16 (KP676759.1) 15 93 / 92 / 93 Limnococcus limneticus Svet06 (GQ375048.1) 2 100 / 99 / 100 sp. PCC 8806 (AF448077.1) 16 100 / 100 / 100 Cyanobacterium stanieri PCC 7202 (AF132782.1) 17 Synechocystis sp. PCC 6308 (AB039001.1) 18 53 / - / - 100 / 100 / 100 Cryptococcum komarkovaum CCALA 054 (MF072346.1) 19 Cryptococcum brasiliense CCIBt3410 (MF072345.1) Inacoccus carmineus CCIBt3475 (MF072349.1) 20 100 / 100 / 100 Inacoccus carmineus CCIBt3411 (MF072347.1) 21 Inacoccus carmineus CCIBt3418 (MF072348.1) 3 22 99/ 99 / 98 Gloeocapsa sp. KO38CU6 (AB067575.1) 99 / 99 / 99 23 Gloeocapsa sp. KO30D1 (AB067579.1) 66 / 59 / 87 Cyanothece sp. ATCC 51142 (AF132771.1) 99 / 99 / 97 24 Cyanothece sp. WH 8902 (AY620238.1) 25 Crocosphaera watsonii WH 8501 (AY620237.1) 26 95 / 91 / 63 Cyanothece sp. PCC 8801 (AF296873.1) Cyanothece sp. WH 8904 (AY620239.1) 92 / 91 / 79 27 Gloeothece sp. KO68DGA (AB067580.1) 96 / 90 / 88 28 Gloeothece sp. KO11DG (AB067577.1) 99 / 99 / 93 29 Cyanothece sp. SKTU126 (AB067581.1) 30 60 / - / 65 Gloeothece sp. SK40 (AB067576.1) Chroococcus cf. westii CCALA 702 (GQ375044.1) 31 99 / 99 / 99 Chroococcus subviolaceus CCIBt3549 (MF072353.1) 94 / 97 / 97 32 100 / 100 / 100 Chroococcus subviolaceus CCIBt3506 (MF072351.1) 33 51 / - / - Chroococcus turgidus CCIBt3508 (MF072352.1) Chroococcus virescens CCALA 701 (GQ375046.1) 34 91 / 88 / 65 Chroococcus prescotii JJCV (AM710385.1) 99 / 99 / 96 35 Chroococcus minutus SAG 41.79 (KM019988.1) 90 / 85 / 94 4 36 Chroococcus minutus CCALA 055 (GQ375047.1) 37 Microcystis aeruginosa PCC 7941 (AJ133171.1) 38 glauca 0BB39S01 (AJ781044.1) 82 / 86 / 69 Synechocystis sp. PCC 6805 (AB041938.1) 96 / 97 / 97 39 64 / 70 / 82Synechocystis sp. PCC 6714 (AB041937.1) 40 86 / 82 / - 100 / 100 / 99 Synechocystis sp. PCC 6702 (AB041936.1) 41 Synechocystis sp. PCC 6803 (AY224195.1) Merismopedia glauca B1448-1 (X94705.1) 55 / - / - 42 naegeliana 0LE35S01 (AJ781043.1) 43 58 / 66 / 52 Snowella litoralis 1LT47S05 (AJ781041.1) 44 100 / 100 / 100 Snowella rosea 1LM40S01 (AJ781042.1) 45 81 / 72 / - Snowella litoralis 0TU37S04 (AJ781040.1) 100 / 99 / 99 Snowella litoralis 0TU35S07 (AJ781039.1) 46 Synechococcus sp. PCC 7502 (AF448080.1) 47 Merismopedia tenuissima 0BB46S01 (AJ639891.1) 48 100 / 100 / 100 Merismopedia sp. CENA106 (EF088332.1) 49 Gloeobacter violaceus PCC 7421 (AF132790.1) 50 0.050 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig. 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Cyanobacteria sp. YW-2019b strain TP201716.4 clone 06 (MN215485.1) 17 Chroococcus sp. CHAB TP201716.4 clone 06 (MT488165.1) 18 Cyanobacteria sp. YW-2019b strain TP201716.5 clone 01 (MN215486.1) 100 / 100 / 100 Chroococcus sp. CHAB TP201716.5 clone 05 (MT488168.1) 19 Cyanobacteria sp. YW-2019b strain TP201716.1 clone 03 (MN215477.1) 20 100 / 100 / 100 Cyanobacteria sp. YW-2019b strain TP201716.3 clone 05 (MN215481.1) 21 Chroococcus sp. CHAB TP201716.3 clone 06 (MT488162.1) 22 Pseudo-chroococcus couteii PMC 885.14 (MT985488) 23 Gloeocapsa sp. PCC 73106 (AB039000.1) 24 Cryptococcum brasiliense CCIBt3410 (MF072345.1) 25 55 / 60 / 58 100 / 100 / 100 Cryptococcum komarkovaum CCALA 054 (MF072346.1) 26 Inacoccus carmineus CCIBt3418 (MF072348.1) 27 100 / 100 / 100 Inacoccus carmineus CCIBt3411 (MF072347.1) 28 Inacoccus carmineus CCIBt3475 (MF072349.1) 29 Chroococcus sp. CCALA 702 (GQ375044.1) 30 99 / 100 / 98 Chroococcus subviolaceus CCIBt3506 (MF072351.1) 100 / 100 / 100 31 Chroococcus subviolaceus CCIBt3549 (MF072353.1) 32 93 / 94 / 86 Chroococcus turgidus CCIBt3508 (MF072352.1) 33 92 / 93 / 94 Chroococcus minutus CCALA 055 (GQ375047.1) 34 100 / 100 / 99 Chroococcus minutus SAG 41.79 (KM019988.1) 35 Microcystis aeruginosa PMC 728.11 36 37 0.010 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig. 6 1

2 Chroococcus turgidus CCITB 3508 Chroococcus subviolaceus CCITB Chroococcus subviolaceus CCITB 3 Inacoccus cluster 3506 3549 4 Inacoccus carmineus CCITB 3411 Inacoccus carmineus CCITB 3475 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Chroococcus cluster

22 Pseudo-chroococcus couteii 23 Cryptococcum cluster 24 25

Cryptococcum brasiliense 26 Cryptococcum komarkovaum 3410 054 Cyanobacteria sp. YW-2019b strain TP201716.5 clone 27 01 28

29 Chroococcus 30 06

31 PMC 885.14 CCITB 32 sp. CHAB TP201716.4 clone 33 CCALA 34 35 36 Cluster 37 Chroococcus-like 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Fig. 7 4 5 6 7 8 A B 9 trichome 10 11 trichome 12 13 14 sh 15 16 17 18 19 20 5 µm 21 10 µm 22 23 24 C sh D sh tz 25 26 27 28 29 30 31 t 32 cw 33 cc 34 35 500 nm 36 500 nm 37 38 sh sh 39 E F tz 40 41 42 Grossisement/détail 43 44 t 45 s 46 Dna ? h 47 tz cy 48 t 49 th 50 51 Under progress : Berenice 52 53 600 nm 400 nm 54 0 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig. 8 Leptolyngbya boryana PCC 6306 (EF429290.1) Leptolyngbya boryanum (X84810.1) Leptolyngbya 1 boryana IAM M-101 (AB245143.1) Leptolyngbya 2 boryana UTEX B 485 (AF132793.1) Leptolyngbya 3 boryana NIES-2135 (LC215287.1) Leptolyngbya 99 / 100 / 99 4 boryana UAM 391 (JQ070062.1) 5 Leptolyngbya boryana PMC 883.14 (MT984284) 100 / 100 / 100 6 Leptolyngbya foveolarum (X84808.1) 7 Leptolyngbya crispata SEV4-3-C6 (AY239598.1) 8 Leptolyngbya sp. SEV4-3-C2 (AY239597.1) 77 / 84 / 66 71 / 68 / 58 9 92 / 83 / 87 Leptolyngbya sp. SEV5-3-C2 (AY239592.1) 10 100 / 100 / 100 Leptolyngbya sp. SEV5-3-C28 (AY239595.1) 11 Leptolyngbya antarctica ANT.L67.1 (AY493572.1) 12 100 / 100 / 100 Leptolyngbya antarctica ANT.LWA.1 (AY493604.1) 13 100 /100 / 100 Limnolyngbya circumcreta CHAB4449 (KR697755.1) 14 Limnolyngbya circumcreta CHAB5650 (KR697752.1) 15 Leptolyngbya nostocorum UAM 387 (JQ070063.1) 80 / 78 / 75 16 66 / 75 / - 82 / 86 / - Leptolyngbya sp. Zehnder 1965/U140 (HM018692.1) 17 Leptolyngbya frigida ANT.L53B.2 (AY493576.1) 18 59 / 71 / - 100 / 100 / 99 Stenomitos rutilans HA7619-LM2 (KF417430.1) 19 Stenomitos sp. HA6792-KK3 (MN152980.1) 20 75/ 74 / 82 |Stenomitos sp. PMC 304.07 (GQ859652.1) 21 78 /76 / - 100 / 99 / 99Stenomitos sp. Ru-0-2 (MH688849.1) 22 Limnothrix redekei 165c (AJ505943.1) 23 100 /100 / 100 24 Limnothrix redekei PMC 272.06 (GQ859647.1) Sodaleptolyngbya stromatolithii PMC 866.14 25 100 / 100 / 99 26 Sodaleptolyngbya stromatolithii PMC 867.14 80 / 74 / 82 Toxifilum mysidocida MYSIDO1 (KX375342.1) 27 100 / 99 / 96 28 Leptolyngbya valderiana SABC022801 (KY807918.1) 29 100 / 100 /100 Leptolyngbya mycoidea LEGE 06108 (HQ832942.1) 30 Uncultured Toxifilum sp. (MH492757.1) 31 98 / 77 / 90 Leptolyngbya sp. HBC8 (EU249119.1) 32 97 / 99 / 82 Leptolyngbya sp. HBC1 (EU249120.1) 33 Leptolyngbya sp. PCC 7375 (AF132786.1) Haloleptolyngbya alcalis PMC 891.15 34 90 / 91 / 35 100 88/ 100 /100 Haloleptolyngbya alcalis PMC 892.15 97 / 96 / 74 36 88 / - / 86 Haloleptolyngbya alcalis KR2005/106 (JN712770.1) 37 Haloleptolyngbya elongata PMC 893.15 38 100 / 100 / 100 Haloleptolyngbya elongata PMC 895.15 39 99 / 96 / 98 Nodosilinea sp. CENA322 (KT731143.1) 40 Leptolyngbya sp. Kovacik 1986/5a (EU528669.2) 41 98 / 81 / 87 Nodosilinea sp. PMC 302.07 (GQ859653.1) 42 |Leptolyngbya sp. 0BB19S12 (AJ639895.1) 43 Nodosilinea sp. CENA523 (KF246490.1) 44 Leptolyngbya sp.PCC 7104 (AB039012.1) 90 / 84 / 75 45 59 / 58 / - Leptolyngbya sp. 0BB24S04 (AJ639893.1) 46 Leptolyngbya sp. 0BB32S02 (AJ639894.1) 47 58 /94 / 52 Nodosilinea nodulosa UTEX 2910 (KF307598.1) 48 58 / - / - Leptolyngbya cf. halophila LEGE 06152 (HQ832915.1) 49 Pseudanabaena sp. CAWBG530 (JX088101.1) 50 100 / 100 / 98 Pseudanabaena catenata SAG 1464-1 (KM020005.1) 51 Pseudanabaena sp. PMC 161.02 (GQ859641.1) 52 99 / 98 / 94 100 / 100 /100 53 Pseudanabaena mucicola PMC 269.06 (GQ859643.1) 54 Gloeobacter violaceus PCC 55 56 0.050 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Fig. 9 2 3 4 5 6 7 8 9 10 11 Microcoleus vaginatus PMC 879.14 (MT984285) Microcoleus vaginatus JR6 (KT753320.1) 12 Microcoleus amoenus BW-1 (EU196636.1) 13 Oscillatoria nigro-viridis PCC 7112 (NR_102469.1 ) Oscillatoria amoena SAG 1459-7 (KM019962.1) 14 Oscillatoria amoena CCAP 1459/39 (HF678512.1) 15 Microcoleus vaginatus JR12 (JN230335.1) 16 Microcoleus vaginatus JR6 (KT753320.1) Microcoleus vaginatus PCC 9802 (AF284803.1) 17 Microcoleus vaginatus SAG 2211 (EF654074.1) 18 Microcoleus vaginatus SAG 2502 (MK953007.1) 19 Microcoleus vaginatus A25 (JN230341.1) 80 / 94 / 89 Microcoleus vaginatus CCALA 145 (JN230340.1) 20 Microcoleus vaginatus Luznice (KC633959.1) 21 63 / 51 / 69 Microcoleus autumnalis SAG 35.90 (EF654081.1) 83 / 85 / 88 Microcoleus autumnalis Arct-Ph5 (DQ493873.2) 22 Microcoleus vaginatus CCALA 154 (JN230342.1) 23 99 / 100 / 100 Kamptonema animale INDIA92 (KP221930.1) 24 Kamptonema animale CCALA 771 (KP221932.1) Trichodesmium contortum (AF013028.1) 25 100 / 100 / 100 Trichodesmium erythraeum IMS101 (113473942:3137158-3138645) 26 Oscillatoria sancta PCC 7515 (NR 114511.1) 27 Trichodesmium thiebautii (AF091321.1) Trichodesmium hildebrandtii (AF091322.1) 28 Laspinema lumbricale UTCC 476 (AF218375.1) 29 Laspinema thermale HK S5 clone cl2 (MF360989.1) 97 / 99 / 100 Laspinema thermale HK S5 clone cl4 (MF360991.1) 30 Laspinema thermale HK S3 clone cl1 (MF360981.1) 31 87 / 89 / 79 Phormidium cf. okenii Led-Z (EU196644.1) 32 Laspinema thermale HK S3 clone cl2 (MF360982.1) Laspinema etoshii KR2008/49 (KC014068.1) 33 Laspinema sp. PMC 878.14 (MT984288) 34 87 / 87 / 92Phormidium cf. terebriformis KR2003/25 (AY575936.1) 35 Geitlerinema splendidum CCALA 1004 (KT315929.1) 100 / 100 / 100 Planktothrix agardhii HAB 637 (FJ159128.1) 36 Planktothrix rubescens NIES-1266 (FJ184421.1) 37 100 / 100 / 100Crinalium magnum SAG 34.87 (AB115965.1) Crinalium epipsammum SAG 22.89 (NR 112218.1) 38 Geitlerinema carotinosum P013 (JQ712598.1) 39 100 / 100 / 100Geitlerinema pseudacutissimum P004 (JQ712617.1) 40 90 / 95 / 95Planktothricoides raciborskii NSLA4 (AB045963.1) Planktothricoides raciborskii NIES-207 (AB045960.1) 41 99 / 99 /99 Planktothricoides raciborskii PMC 877.14 (MT984287) 42 Planktothricoides raciborskii OR1-1 (B045964.1) 43 Candidatus Planktothricoides niger OB (KC407687.1) Candidatus Planktothricoides rosea OP (KC407688.1) 44 99 / 100 / 99 Phormidium cf. nigrum CCALA 147 (AM398779.1) 45 Phormidium cf. irriguum CCALA 759 (EU196638.1) 98 / 99 / 99Lyngbya robusta CCALA 966 (JN854138.1) 46 98 / 100 / 99 Lyngbya hieronymusii N-929 (JN854140.1) 47 Lyngbya aestuarii PCC 7419 48 Lyngbya martensiana H3b/33 (JN854141.1) Lyngbya martensiana MBDU 518 (KX913923.1) 99 / 100 / 99 49 Lyngbya martensiana H3b/7 (JN854142.1) 50 Lyngbya martensiana AUS-JR/MT/NT-124 (KX670285.1) 94 / 99 / 98 51 Lyngbya martensiana PMC 880.14 (MT984286) Gloeobacter violaceus PCC 7421 (AF132790.1) 52 53 0.020 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 4 5 Fig. 10 6 7 8 9 10 11 A B 12 13 ac 14 15 16 trichome 17 18 19 20 21 22 23 20 µm 10 µm 24 25 26 C D b 27 28 its 29 trichome mu 30 cm 31 ac 32 33 b cy t 34 35 36 37 38 5 µm ca 1 µm 39 40 41 E cm F 42 its 43 t 44 45 46 cy 47 t 48 ae 49 c 50 51 52 53 0.5 µm 1 µm 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 4 5 Fig. 11 6 7 8 9 A BB 10 11 ac 12 13 14 15 ac 16 17 18 19 20 21 22 10µm 10µm10µm 23 24 25 C D 26 27 28 ca 29 t ab 30 31 32 33 34 ab 35 cw 36 ca 37 1µm 600nm 38 39 40 41 E F 42 cy 43 44 cy 45 46 47 gv 48 49 50 t 51 t 52 53 600nm t 1µm 54 its 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 4 5 6 7 Fig. 12 8 9 10 11 12 13 A B 14 15 ac 16 17 18 19 20 21 f 22 filament 23 24 20µm 1µm 25 26 27 C D 28 t 29 30 cw 31 cw t 32 33 34 35 36 37 38 39 2µm cm cy 1µm 40 cy 41 42 E F cw ac 43 cw t 44 45 t 46 47 48 49 cy gv 50 51 52 a 53 1µm b 54 400nm 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 4 Fig. 13 5 6 7 8 9 A B 10 11 12 13 14 f 15 16 b 17 18 sh f 19 20 21 f 22 23 10µm 1µm1µm 24 25 26 C cy D 27 28 sh 29 its 30 C gv 31 t 32 ca 33 mu 34 35 36 cw 37 cw 38 1µm 2µm 39 40 41 42 E cw F 43 gv 44 t 45 gv its 46 ca c cw 47 48 49 50 ab pbs 51 52 53 t 54 600nm 400nm 55 its 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Fig. 14 4 5 6 7 8 9 100 / 98 / 99 Trichormus variabilis GREIFSWALD (AJ630457.1) 70 / 73 / - Trichormus variabilis HINDAK 2001/4 (AJ630456.1) 10 Anabaena cylindrica PCC 7122 11 Nodularia spumigena CCY9414 100 / 100 / 98 Nodularia sp. PCC 73104 12 Cuspidothrix issatschenkoi 0tu37s7 82 / 75 / - Anabaena sp. 90 13 88 / 88 / 96 Dolichospermum solitarium PMC 196.03 14 97 / 98 / 97 Dolichospermum flos-aquae PMC 207.03 74 / 70 / 69 Nostoc azollae 0708 15 Sphaerospermopsis reniformis 07 01 100 / 100 / 100 Sphaerospermopsis aphanizomnoides 22F6 1 16 99 / 93 / 96 Aliinostoc morphoplasticum NOS (KY403996.1) 17 57 / 69 / 62 Aliinostoc sp. CENA548 (KX458492.1) 99 / 99 / 97 Aliinostoc catenatum SA24 (MK503792.1) 18 Aliinostoc magnkinetifex SA18 (MK503791.1) 54 / 60 / - 19 Aliinostoc soli ZH1(3) PS (MH497065.1) 99 / 99 / 99 Aliinostoc sp. PMC 882.14 (MT984291) 20 Aliinostoc constrictum SA30 (MK503793.1) 83 / 59 / 89 Aliinostoc tiwarii LI PS (MH497064.1) 21 100 / 100 / 100 Minunostoc cylindricum CHAB 5843 clone 3 (MK045297.1) 70 / 53 / - Minunostoc cylindricum CHAB 5844-1 (MK045295.1) 86 / 86 / - 22 Desikacharya constricta S10 (MK354274.1) 23 81 / 71 / - Desikacharya sp. SAG 2306 (GQ287649.1) 99 / 100 / 99 Nostoc sp. PCC 6720 (DQ185240.1) 24 97 / 97 / - Nostoc sp. PCC 7107 25 Trichormus azollae Kom-BAI 1983 93 / 95 / 92 Desikacharya thermotolerans 9C-PS (KX2526751) 26 Nostoc sp. PCC 9426 (AM711538.1) Trichormus doliolum 1 27 Nostoc sp. PCC 7524 68 / 60 / - Trichormus variabilis ATCC 29413 (HF678501.1) 28 100 / 95 / 100Nostoc sp. PCC 7120 29 Nostoc flagelliforme CCNUN1 (NZ_CP024785.1) 56 / 59 / 65 Nostoc punctiforme PCC 73102 30 Nostoc sp. 1tu14s8 (AJ630453.1) 31 100 / 100 / 99 AF0276541__Nostoc sp. PCC 9709 (AF027654.1) Nostoc calcicola III 32 Nostoc edaphicum X Nostoc sp. ATCC53789 (AF062638.1) 33 Nostoc sp. PMC 881.14 (MT984289) 34 Nostoc ellipsosporum V Desmonostoc muscorum Lukesova_1/87 (AM711523.1) 35 99 / 99 / 92Desmonostoc muscorum I (AJ630451.1) 36 Desmonostoc muscorum II 93 / 92 / 99 Desmonostoc sp. PCC 6302 (HG004582.1) 37 Desmonostoc sp. PCC 7422 (HG004586.1) 99 / 92 / 86 Nostoc linckia NIES-25 (NZ_AP018222.1) 38 Desmonostoc sp. PCC 8306 (HG004584.1) 100 / 100 / 100 Desmonostoc sp. OSNI32S01 (HG004587.1) 39 74 / 73 / 92Desmonostoc sp. PCC 9230 (HG004585.1) 40 Rivularia sp. PCC 7116 74 / 73 / - Fischerella muscicola SAG1427 1 41 100 / 100 / 100Hapalosiphon sp. IAM M 264 Calothrix sp. PCC 7103 (AM230700.1) 83 / 67 / - 42 Calothrix parietina CCAP 1410/10 (HF678479.1) 43 100 / 100 / 99 Calothrix desertica PCC 7102 (NR 114995.1) Calothrix sp. PMC 884.14 (MT984290) 44 100 / 100 / 95Calothrix thermalis PCC 7715 (AM230701.1) Calothrix sp. PCC 8909 (AM230693.1) 45 100 / 100 / 100 Calothrix sp. AHLA9 (AM230694.1) 70 / 79 / - 46 Calothrix sp. PCC 6303 Calothrix sp. XP11C (AM230698.1) 47 Macrochaete lichenoides SAG 32.92 (KU559618.1) Macrochaete psychrophila TS40B/09 (KR350574.1) 68 / 73 / - 48 Macrochaete psychrophila CCALA 1092 (KR350577.2) 100 / 100 / 100 49 Macrochaete psychrophila ANT SPH 13/96 (KT336438.2) 84 / 80 / 94Macrochaete psychrophila CCALA 32 (KT336439.2) 50 Gloeobacter violaceus PCC 7421 (NR 074282.1)

51 0.050 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 4 5 Fig. 15 6 7 8 9 10 11 A C 12 13 14 15 16 17 f cp 18 19 20 B sh 21 22 cp

23 20µm 24 25 26 D E sh 27 28 29 sh cw 30 cw 31 32 33 34 35 mu 36 37 mu 38 1µm 1µm 39 40 41 F G 42 43 t 44 sh 45 46 t t 47 48 49 cw t t 50 cw 51 52 500nm 200nm 53 mu 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 4 Fig. 16 5 6 7 8 9 10 B B 11 A 12 A B 13 14 15 16 17 b 18 19 20 21 22 23 20µm 1µm 24 1µm 25 26 C D 27 cw 28 29 30 31 ca 32 33 34 35 36 37 38 500 nm 2µm 39 40 41 E cw F 42 cw 43 44 45 46 t 47 48 49 50 cif 51 52 53 500 nm 500 nm 54 55 500 nm 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 4 5 6 Fig. 17 7 8 9 10 11 12 A B 13 14 15 sh 16 17 18 A 19 20 21 22 23 20µm 5 µm 24 25 sh 26 C D 27 28 29 30 31 32 mu 33 sh 34 cw 35 36 ca 37 4µm 800 nm 38 39 40 E sh F ld cw 41 42 cw 43 44 45 46 47 48 ca 49 50 51 52 800 nm 800 nm 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Table 1. List of cyanobacteria strains isolated from the retention basin of Thermes of 4 Balaruc-Les-Bains and their corresponding strain numbers, habitat (water column or biofilms) 5 6 and sampling dates. PMC: Paris Museum Collection. 7 8 Order Species Strain number Habitat Sampling date 9 Chroococcales Pseudo-chroococcus couteii PMC 885.14 Epilithic biofilm 23/06/2014 10 Synechococcales Leptolyngbya boryana PMC 883.14 Mud surface 12/05/2014 11 12 Oscillatoriales Planktothricoides raciborskii PMC 877.14 Mud surface 26/05/2014 13 Laspinema sp. PMC 878.14 Mud surface 23/06/2014 14 15 Microcoleus vaginatus PMC 879.14 Epilithic biofilm 26/05/2014 16 Lyngbya martensiana PMC 880.14 Epilithic biofilm 23/06/2014 17 18 Nostocales Nostoc sp. PMC 881.14 Epilithic biofilm 23/06/2014 19 Aliinostoc sp. PMC 882.14 Epilithic biofilm 23/06/2014 20 Calothrix sp. PMC 884.14 Mud surface 12/05/2014 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Supplementary materials: 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Figure S1. Pigments composition (%) of the nine studied strains, isolated from Thermes de 41 Balaruc-Les-Bains. PMC: Paris Museum Collection, Museum National of Natural History. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 69 / - / 67 Nostoc flagelliforme CCNUN1 (NZ CP0247851:81819-83309) 3 Nostoc punctiforme PCC 73102 100 / - / 92 Nostoc sp. PCC 9709 (AF027654.1) Nostoc calcicola 4 53 / 95 / 95 III Nostoc sp. ATCC 53789 (AF062638.1) 51 / - / - Nostoc edaphicum X Nostoc sp. PMC 881.14 (MT984289) 5 50 / - / - Nostoc ellipsosporum V 100 / 100 / 95 Nostoc muscorum Lukesova 1/87 (AM711523.1) 6 Desmonostoc muscorum I (AJ630451.1) 94 / 69 / 78 Desmonostoc sp. PCC 7422 (HG004586.1) 98 / 94 / 98 Desmonostoc sp. PCC 9230 (HG004585.1) 7 Nostoc linckia NIES-25 (NZ AP0182221:c3653887-3652392) 100 / 100 / 100 Trichormus variabilis ATCC 29413 (HF678501.1) 86 / 50 / - Nostoc sp. PCC 7120 8 Nostoc sp. PCC 7524 Trichormus doliolum 1 9 99 / 91 / 99 Nostoc sp. PCC 9426 (AM711538.1) Trichormus azollae Kom-BAI/1983 76 / 79 / 87 100 / 97 / 99 Nostoc sp. PCC 6720 (DQ185240.1) 10 Nostoc sp. PCC 7107 84 / 99 / - 55 / 53 / 84 Minunostoc cylindricum CHAB 5844-1 (MK045295.1) 100 / 100 / 100 Minunostoc cylindricum CHAB 5843 clone 3 (MK045297.1) 11 83 / 93 / 84 Aliinostoc tiwarii LI PS (MH497064.1) 53 / 85 / 81 Aliinostoc constrictum SA30 (MK503793.1) Nostocales 12 99 / 98 / 99 Aliinostoc sp. PMC 882.14 (MT984291) 59 / 69 / 59 Aliinostoc soli ZH1(3) PS (MH497065.1) Aliinostoc magnkinetifex SA18 (MK503791.1) 13 99 / 93 / 93 Aliinostoc catenatum SA24 (MK503792.1) 69 / 84 / 72 Aliinostoc morphoplasticum NOS (KY403996.1) 97 / 100 / 95 Aliinostoc sp. CENA548 (KX458492.1) 14 Anabaena sp. 90 96 /9 7 / 94 Anabaena tenericaulis 08 10 99 / 80 / 98 15 82 / 55 / 73 Dolichospermum solitarium PMC 196.03 94 / 95 / 96 Dolichospermum flos-aquae PMC 207.03 Cuspidothrix issatschenkoi 0tu37s7 16 100 / 99 / 99 Trichormus variabilis HINDAK 2001/4 (AJ630456.1) 61 / - / - Trichormus variabilis GREIFSWALD (AJ630457.1) Anabaena cylindrica PCC 7122 17 Anabaena bergii PMC 314.07 63 / 81 / 78 Nodularia sp. PCC 73104 18 99 / 91 / 99 Nodularia spumigena CCY9414 Calothrix sp. PCC 6303 100 / 100 / 100 Calothrix sp. PCC 8909 (AM230693.1) 19 Calothrix sp. XP11C (AM230698.1) 85 / 95 / - 100 / 100 / 100 Calothrix sp. PMC 884.14 (MT984290) 20 ((MT Calothrix thermalis PCC 7715 (AM230701.1) 100 / 100 / 100 Calothrix desertica PCC 7102 (NR 114995.1) 91 / 79 / 69 Calothrix sp. PCC 7103 (AM230700.1) 21 Calothrix parietina CCAP 1410/10 (HF678479.1) Leptolyngbya boryanum (X84810.1) Leptolyngbya boryana PCC 6306 (EF429290.1) 22 99 / 100 / 99 Leptolyngbya boryana IAM M-101 (AB245143.1) Leptolyngbya boryana NIES-2135 (LC215287.1) 100 / 100 / 100 23 Leptolyngbya boryana PMC 883.14 (MT984284) Leptolyngbya crispata SEV4-3-C6 (AY239598.1) 67 / 87 / - 86 / 95 / 79 Leptolyngbya sp. SEV5-3-C2 (AY239592.1) 24 100 / 100 / 100 Leptolyngbya sp. SEV5-3-C28 (AY239595.1) Leptolyngbya antarctica ANT.L67.1 (AY493572.1) 100 / 100 / 100 Leptolyngbya antarctica ANT.LWA.1 (AY493604.1) 25 100 / 100 / 99 Stenomitos sp. PMC 304.07 (GQ859652.1) 62 / 72 /65 Stenomitos sp. Ru-0-2 (MH688849.1) 26 Stenomitos rutilans HA7619-LM2 (KF417430.1) Synechococcales 100 / 100 / 100 Leptolyngbya frigida ANT.L53B.2 (AY493576.1) 92 Leptolyngbya nostocorum UAM 387 (JQ070063.1) 27 87 Leptolyngbya sp. Zehnder 1965/U140 (HM018692.1) 100 / 100 / 100 Leptolyngbya mycoidea LEGE 06108 (HQ832942.1) 50 / - / - Leptolyngbya valderiana SABC022801 (KY807918.1) 28 Limnothrix redekei 165c (AJ505943.1) 100 / 100 / 100 Limnothrix redekei PMC 272.06 (GQ859647.1) 29 Nodosilinea sp. CENA322 (KT731143.1) 100 / 100 / 100 Nodosilinea nodulosa UTEX 2910 (KF307598.1) 87 / 64 / 69 Nodosilinea sp. PMC 302.07 (GQ859653.1) 30 95 / 90 / 63 Nodosilinea sp. CENA523 (KF246490.1) 100 / 100 / 100 Synechocystis sp. PCC 6702 (AB041936.1) 31 Synechocystis sp. PCC 6803 (AY224195.1) 96 / 66 / 97 Cyanothece sp. ATCC 51142 (AF132771.1) 100 / 100 / 99 Gloeocapsa sp. KO30D1 (AB067579.1) 32 95 / 94 / 87 Cyanothece sp. WH 8902 (AY620238.1) 99 / 93 / 89 Gloeothece sp. KO11DG (AB067577.1) 100 / 100 /100 Gloeothece sp. SK40 (AB067576.1) 33 Cyanothece sp. PCC 8801 (AF296873.1) Cyanobacteria sp. YW-2019b strain TP201716.4 clone 06 34 100 / 100 / 100 (MN215485.1) Cyanobacteria sp. YW-2019b strain TP201716.1 clone 03 (MN215477.1)Pseudo-chroococcus couteii PMC 885.14 (MT985488) Inacoccus carmineus CCIBt3475 35 100 / 100 / 100 (MF072349.1) Inacoccus carmineus CCIBt3418 Chroochococcales (MF072348.1) Gloeocapsa sp. PCC 73106 100 / 100 / 100 (AB039000.1) Uncultured Limnococcus sp. Alla11otu7-1 16 (KP676759.1) 36 98 / 99 / 76 Limnococcus limneticus Svet06 100 / 100 / 100 Cryptococcum(GQ375048.1) komarkovaum CCALA 054 37 Cryptococcum(MF072346.1) brasiliense CCIBt3410 100 / 100 / 99 Chroococcus(MF072345.1) minutus SAG 41.79 83 / 51 / - (KM019988.1) Chroococcus minutus CCALA 055 38 (GQ375047.1) Chroococcus turgidus CCIBt3508 100 / 100 / 100 (MF072352.1) Chroococcus cf. westii CCALA 702 65 / - / - Chroococcus(GQ375044.1) subviolaceus CCIBt3549 39 99 / 96 / 91 Chroococcus(MF072353.1) subviolaceus CCIBt3506 100 / 100 / 100 Phormidium cf. nigrum (MF072351.1)CCALA 147 40 (AM398779.1) Phormidium cf. irriguum CCALA 759 (EU196638.1) Lyngbya martensiana H3b/7 100 Lyngbya(JN854142.1) martensiana PMC 880.14 (MT984286) 41 100 / 99 / 99 Trichodesmium thiebautii 100 /100 / 100 (AF091321.1) Trichodesmium hildebrandtii 42 (AF091322.1) Trichodesmium erythraeum IMS101 (113473942:3137158- 100 / 100 / 100 Kamptonema3138645) animale INDIA92 62 / - / 51 (KP221930.1)Kamptonema animale CCALA 771 43 Microcoleus(KP221932.1) vaginatus Luznice (KC633959.1) 77 /51 / - Microcoleus autumnalis Arct-Ph5 (DQ493873.2) Microcoleus autumnalis SAG 35.90 (EF654081.1) 44 Microcoleus vaginatus CCALA 154 (JN230342.1) 100 / 99 / 99 Microcoleus vaginatus PCC 9802 (AF284803.1) 45 Microcoleus vaginatus SAG 2502 (MK953007.1) Microcoleus vaginatus PMC 879.14 (MT984285) Oscillatoriales 81 / 81 / 60 Microcoleus vaginatus JR12 (JN230335.1) 46 Microcoleus vaginatus JR6 (JN230339.1) 100 / 100 / 99 Lyngbya robusta CCALA 966 (JN854138.1) Lyngbya aestuarii PCC 7419 47 Planktothricoides raciborskii NSLA4 (AB045963.1) Planktothricoides raciborskii PMC 877.14 (MT984287) 48 100 / 100 / 100 Planktothricoides raciborskii NIES-207 (AB045960.1) 52 / 62 / - Planktothricoides raciborskii OR1-1 (B045964.1) Laspinema lumbricale UTCC 476 (AF218375.1) 49 Laspinema thermale HK S5 clone cl2 (MF360989.1) 100 / 100 /100 Laspinema thermale HK S3 clone cl2 (MF360982.1) 81 / 91 /68 Laspinema sp. PMC 878.14 (MT984288) 50 Phormidium cf. terebriformis KR2003/25 (AY575936.1) 90 / - / 86 Laspinema etoshii KR2008/49 (KC014068.1) 0.050 51 Gloeobacter violaceus PCC 7421 (AF132790.1) 52 53 Figure S2. Consensus phylogenetic tree based on 16S rRNA gene sequences of representative 54 cyanobacteria strains (129 sequences, 1446 aligned nucleotide positions, GTR+G+I model) 55 belonging to the orders Oscillatoriales, Nostocales, Chroococcales, Synechococcales and the 56 studied strains form Thermes de Balaruc-Les-Bains (in bold). Gloeobacter violaceus was 57 used as outgroup. Numbers above branches indicate bootstrap support (>50%) from 1000 58 replicates. Bootstrap values are given in the following order: maximum likelihood / neighbor 59 60 joining / maximum parsimony.

2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Table S1. List of GenBank accession numbers. PMC: Paris Museum Collection. 4 5 Order Species Strain number 16S 16S -23S 6 7 Chroococcales Pseudo-chroococcus couteii PMC 885.14 - MT985488 8 Synechococcales Leptolyngbya boryana PMC 883.14 MT984284 - 9 10 Oscillatoriales Planktothricoides raciborskii PMC 877.14 MT984287 - 11 Laspinema sp. PMC 878.14 MT984288 - 12 Microcoleus vaginatus PMC 879.14 MT984285 - 13 14 Lyngbya martensiana PMC 880.14 MT984286 - 15 Nostocales Nostoc sp. PMC 881.14 MT984289 - 16 17 Aliinostoc sp. PMC 882.14 MT984291 - 18 Calothrix sp. PMC 884.14 MT984290 - 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Table S2. Comparison of the morphological features of Pseudo-chroococcus couteii PMC 885.14 strain with other Chroococcales (Gleocapsa, 4 Limnococcus, Inacoccus, Cryptococcum) genera (Fig. 4). nd: not determined. 5 6 Taxonomic assignment Strain Origin Habitat Vegetative cell Reference 7 8 Shape Sheath Color Size min.-max. (mean), µm 9 Pseudo-chroococcus couteii PMC 885.14 France Epilithic biofilm More or less spherical, Colorless, slightly Pale green, 14-17.8 (15.6) This study 10 oval, hemispherical after lamellate blue-green 11 division or grey 12 Cyanobacteria sp. YW-2019b TP201716.1 China nd nd nd nd nd Institute of 13 clone 03 Hydrobiology site (MN215477.1) 14 web 15 Gleocapsa sp. PCC 73106 Switzerland Sphagnum bog nd nd nd nd PCC site web 16 (AB039000.1) collection 17 Limnococcus limneticus Svet06 Czech Rep Fishpond Svet nd nd nd 7-11 Komărkovă et al. 2010 (GQ375048.1) plankton 18 Inacoccus carmineus sp. nov. CCIBt3411 Brazil (Santa Terrestrial, on Hemispherical to rounded Hyaline to intense Purple brownish 4.7–11.9 (6.7) Gama et al. 2019 19 (MF072347.1) Virgínia Park) concrete. red-colored smooth to green- 20 or rarely lamellate brownish 21 Inacoccus carmineus sp. nov. CCIBt3475 Brazil (Ilha do Wet Rock Hemispherical to rounded Hyaline to intense Purple brownish 4.7–11.9 (6.7) Gama et al. 2019 22 (MF072349.1) Cardoso State red-colored smooth to green- 23 Park) or rarely lamellate brownish 24 25 Cryptococcum komarkovaum CCALA 054 Unknown Aquatic, thermal Hemispherical to rounded Hyaline, smooth or Blue-green to 8.7–13.9 Gama et al. 2019 26 (MF072346.1) spring solitary or rarely grouped rarely lamellate yellowish-green (10.8) 27 in few-celled colonies 28 29 Cryptococcum brasiliense CCIBt3410 Brazil (Ilha do Terrestrial, on dry Hemispherical to rounded Hyaline, smooth or Green to blue- 8.3–15.5 Gama et al. 2019 30 (MF072345.1) Cardoso State soil among pebbles solitary or rarely grouped rarely lamellate green 31 Park) in few-celled colonies 32 33 34 35 36 37 38 39 40 41 42 43 4 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Table S3. Comparison of the morphological features of the Leptolyngbya boryana sp. PMC 883.14 strain with other Leptolyngbya species 4 belonging to the same phylogenetical cluster (Fig. 8). nd: not determined. 5 6 Taxonomic Strain Origin Habitat Trichome Vegetative cell Reference 7 assignment 8 Color Sheath Shape Shape of Width min.- Shape Width min.- Length min.- 9 terminal cell max. (mean) max. max. µm (mean), µm (mean), µm 10 11 Leptolyngbya PMC 883.14 France Freshwater, Pale to Thin, colourless, Straight, curved, Rounded 1.5-2.4 Moniliform, 1.5-2.4 (2.1 0.9 - 2 This study 12 boryana benthic, bright attached to the distinctly hemispherical shorter than ±0.24) (1.52 13 biofilms blue- trichome constricted, wide length:width ±0.27) green ratio = 0.75 14 15 Leptolyngbya PCC 6306 nd Freshwater, Blue- Absent Long, straight or Rounded nd More or less length:width length:wid Chakraborty 16 boryana (EF429290.1) metaphytic, green wavy, distinctly isodiametric ratio = 0.78 th ratio = et al. 2019 lentic constricted, false 0.78 17 branching 18 19 Leptolyngbya UAM 391 Spain Freshwater, Intense Hyaline, Strongly Rounded nd nd 2.1–3.2 1.2–3.6 Loza et a. 20 boryana (JQ070062.1) Guadarrama benthic, blue- homogeneous constricted, false 2013 21 river biofilms green or sometimes branching 22 pale color thick, separated 23 from the filament 24 25 Leptolyngbya IAM M-101 Japan Freshwater nd nd nd nd nd nd nd nd nd 26 boryana (AB245143.1 27 ) 28 Leptolyngbya NIES-2135 Japan Freshwater nd nd nd nd nd nd nd nd nd 29 boryana (LC215287.1) 30 Leptolyngbya 1964/112 nd nd nd Presence of a Constrictions at nd nd nd 3.8 3.8 Nelissen et 31 foveolarum (X84808.1) sheath the cross-walls al. 1996 32 Leptolyngbya PCC 73110 nd Freshwater nd Variable Constrictions at nd nd nd 1.9 – 2.6 1.9 – 2.9 Nelissen et 33 boryanum (X84810.1) the cross-walls al. 1996 34 35 36 37 38 39 40 41 42 43 5 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

FEMS Microbiology Ecology Page 48 of 57

1 2 3 Table S4. Comparison of the morphological features of the Planktothricoides raciborskii PMC 877.14 strain with other Planktothricoides 4 species belonging to the same phylogenetical cluster (Fig. 9). nd: not determined. 5 6 Taxonomic assignment Strain Origin Habitat Trichome Vegetative cell Reference 7 Color Sheat Shape Shape of Width min.- Shape Width min.- Length min.- 8 terminal max. (mean) max. max. 9 cell µm (mean), µm (mean), µm 10 Planktothricoides PMC 877.14 France Freshwater, Pale- Thin, Straight Rounded 5-7.3 (2020) Cylindrical, 5-7.3 (6 3.3-5.4 (4.48 This study 11 raciborskii mud green, colourless +/- curved conical shorter than ±0.56) ±0.46) 12 brown wide Candidatus OP Guadeloupe Marine Pink Thin, clear Straight Slightly 5–10 Disk-shaped 5–10 1.25-2.5 Guidi- 13 Planktothricoides rosea (KC407688.1) mangrove slightly rounded greater in Rontani et 14 Mat on waved without width than in al. 2014 15 thesediment calyptra length from the 16 periphyton 17 18 Candidatus OB Guadeloupe Marine Black Thin, clear Straight Slightly 5–10 Disk-shaped 5–10 1.25-2.5 Guidi- 19 Planktothricoides niger (KC407687.1) mangrove slightly rounded greater in Rontani et 20 Mat on the waved without width than in al. 2014 sediment calyptra length 21 from the 22 periphyton 23 Planktothricoides OR1-1 Thailand Freshwater Pale blue- nd Straight Rounded nd nd 5.4–2.2 (Group IV) : Suda et 24 raciborskii (B045964.1) Bangkok planktic in green attenuated without (Group IV) : 1.7–6.7 al. 2002 lakes yellow- at the calyptra 5.4–12.2 (3.3 ± 0.6) 25 green ends 26 Planktothricoides NSLA4 Australia Freshwater nd nd Straight nd nd nd (Group IV) (Group IV) : Suda et 27 raciborskii (AB045963.1) New South planktic in attenuated 5.4–12.2 1.7–6.7 al. 2002 28 Wales Lake lakes at the 29 Alexandrina ends 30 Planktothricoides NIES-207 Japan Ibaraki Freshwater Pale blue- nd Straight Tapered, nd nd 5.4–9.8 (Group IV) : Suda et 31 raciborskii (AB045960.1) Lake planktic in green attenuated bent (Group IV) 1.7 –6.7 al. 2002 32 Kasumigaura lakes yellow- at the 5.4–12.2 33 green ends 34 Planktothricoides sp. SR001 Singapore Freshwater, nd nd Straight Attenuated nd Cylindrical, (8.13 ±0.92 5.48 (±2.01) Te et al. 35 (NZ_LIUQ010 planktic attenuated without shorter than width: 2017 36 00113.1) reservoir at the calyptra wide length ratio 37 ends 1.68 (± 0.65) 38 39 40 41 42 43 6 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Table S5. Comparison of the morphological features of the Laspinema sp. PMC 878.14 strain with other Laspinema and Phormidium species 4 belonging to the same phylogenetical cluster (Fig. 9). nd: not determined. 5 6 Taxonomic Strain Origin Habitat Trichome Vegetative cell Reference 7 assignment 8 Color Sheath Shape Shape of Width min.- Shape Width min.- Length min.- 9 terminal cell max. (mean) max. (mean), max. (mean), µm µm µm 10 11 Laspinema sp. PMC 878.14 France Benthic on Bright Lacking Straight, nutated Obtuse-conical, 3.8 - 4.8 Shorter than 3.8 - 4.8 2.3 - 3.3 This study mud blue- sheath at the end rounded-conical, wide (4.26 ±0.27) (2.87 ±0.28) 12 freshwater, green without calyptra 13 brackish 14 waters 15 Laspinema HK S5 clone 2 Iran, Thermal Blue- Thin, Straight in natural Rounded, 3 – 4 (5) Shorter than 3 – 4 (5) 1 – 2,4 Heidari et 16 thermale Mahallat spring green or inconspicuo environment conical, shortly wide al. 2018 17 (MF360989.1) olive us forming benthic bent green mats, slightly 18 constricted on 19 cross walls 20 Laspinema UTCC 476 Antarctica Saline pond nd nd nd Rounded, nd nd 5 – 6 (7) 2 - 4 Casamatta 21 lumbricale (AF218375.1) on McMurdo straight et al. 2005 22 Ice Shelf 23 Laspinema KR2008/49 Namibia Hyposaline Pale Hyaline, Solitary, straight Round, conical; nd Mainly wider 5.5 ±1.5 3.0 ±1.0 Dadheech 24 etoshii (KC014068.1) Etosha water bright colorless or curvy, not or straight or bent than long et al. 2013 National puddles blue- slightly without calyptra 25 Park, springs and green constricted 26 Okandeka wet soil 27 Spring 28 Phormidium cf. Led-Z Czech a pond, Dark Delicate Gradually Bent, elongated nd Slightly 5.6-6.5 (3.7- nd Lokmer okenii (EU196644.1) Republ benthos blue- colourless narrowed towards usually rounded shorter than 7.5 ; 6.1) 2007 29 Lednice na green end, distinctly wide or cell 30 Moravě park constricted at isodiametrical, length/width 31 cross-walls sometimes ratio 0.5-0.8 longer than 32 wide 33 Phormidium cf. KR2003/25 Kenya, Hot Hyposaline nd nd Solitary, straight Rounded nd Mainly 5.5 ±0.5 3.0 ±1.0 Dadheech 34 terebreformis (AY575936.1) spring, Lake hot springs to undulating without calyptra isodiametric et al. 2013 Bogoria, Constricted 35 36 37 38 39 40 41 42 43 7 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Table S6. Comparison of the morphological features of the Microcoleus vaginatus PMC 879.14 strain with other Microcoleus species or strains 4 belonging to the same phylogenetical cluster (Fig. 9). nd: not determined. 5 6 Taxonomic Strain Origin Habitat Trichome Vegetative cell Reference 7 assignment 8 Color Sheath Shape Shape of Width min.- Shape Width min.- Length min.- 9 terminal cell max. (mean) max. (mean), max. (mean), µm µm µm 10 11 Microcoleus PMC 879.14 France Epilithic biofilm Bright blue Thin, firm Straight, Elongated, capitate, 4.7 - 6.2 Shorter than 4.7 - 6.2 2.4 - 4.8 this study vaginatus green cylindrical with rounded wide (5.63 ± 0,36) (3.68 ± 0.52) 12 or truncated calyptra 5 (2014) 2 (2014) 13 Microcoleus SAG 35.90 Switzerland, Freshwater nd nd nd nd nd nd nd nd SAG site 14 autumnalis (EF654081.1) brook in web 15 Verzascatal collection 16 Microcoleus Arct-Ph5 Canada, Mineral soil nd Few Single filaments Rounded calyptra 8 – 9 nd 3 – 4 2 – 4 Elster et al. 17 autumnalis (DQ493873.2) Ellesmere filaments or mucilaginous 1997 18 Island possessed colonies 19 Microcoleus CCALA 154 Japan, East Cooling tower nd nd nd nd nd nd nd nd Strunecky 20 vaginatus (JN230342.1) Asia et al. 2013 21 Microcoleus JR12 Antarctica Gray-black mats nd nd nd nd nd nd nd nd Strunecky 22 vaginatus (JN230335.1) James Ross biofilm on rocks et al. 2013 23 Island 24 Microcoleus JR6 Antarctica Periphyton in nd nd nd nd nd nd nd nd Strunecky 25 vaginatus (JN230335.1) James Ross wetlands close et al. 2013 26 Island Lachman Lake 27 Microcoleus A25 Tennessee, Soil nd nd nd nd nd nd nd nd Strunecky 28 vaginatus (JN230341.1) North America et al. 2013 29 Oscillatoria SAG 1459-7 Germany, Bot. Freshwater nd nd nd nd nd nd nd nd SAG site 30 amoena (KM0119962. Gard. Univ. web 31 1) Bonn collection Microcoleus Cosmopolitan Subaerophytic, Bright blue Colourless, Trichome long not Capitate, bluntly (2,5) 3 - 7 Shorter than 2 - 5 (6,7) nd Komárek & 32 vaginatus on soils, stones, green or constricted rounded, with conical, (9?) wide Anagnostidi 33 dried mud, olive green obtuse-conical or s 2005 34 waterfalls, littoral to dirty hemispherical calyptra of lakes, rarely green 35 submersed in 36 stagnant and 37 flowing waters 38 39 40 41 42 43 8 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Table S7. Comparison of the morphological features of the Lyngbya martensiana PMC 880.14 strain with other Lyngbya martensiana belonging 4 to the same phylogenetical cluster (Fig. 9). nd: not determined. 5 6 Taxonomic Strain Origin Habitat Trichome Vegetative cell Reference 7 assignment 8 Color Sheath Shape Shape of Width min.- Shape Width min.- Length min.- 9 terminal cell max. (mean) max. (mean), max. (mean), µm µm µm 10 11 Lyngbya PMC 880.14 France Epilithic Pale blue- Thick, Cylindrical Widely rounded, 4.8 - 6.6 Shorter than 4.8 - 6.6 (5.3 1.2 - 2.1 This study martensiana biofilm green, colourless straight, flexuous hemispherical wide ± 0.47) (1.6 ± 0.2) 12 olive without calyptra 13 green 14 Lyngbya AUS- India Rice Field Blue Colourless, Entangled and Rounded without 6-10 Cells ½ -1/4 1.5 – 2.5 Thajamambi 15 martensiana JR/MT/NT- soil green firm, thick interwoven, long calyptra, not times long as et al. 2016 124 flexible, straight capitate broad 16 (KX670285.1) 17 Lyngbya MBDU 518 India Marine nd nd nd nd nd nd nd nd Genbank site 18 martensiana (KX913923.1) web 19 Lyngbya H3b/7 Guatemala, Periphytic Bright Firm, Cylindrical, Not attenuated 9 nd (6.2) 7 – 7.5 1.4 Komárek et 20 martensiana (JN854142.1) Lake Atilan freshwater blue smooth straight or slightly (8.7) al. 2013 21 green from bent, coiled, or Turicchia et outside screw-like coiled al. 2009 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 9 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Table S8. Comparison of the morphological features of the Nostoc sp. PMC 881.14 strain with other Nostoc species belonging to the same 4 phylogenetical cluster (Fig. 14). nd: not determined. 5 6 Taxonomic Strain Origin Habitat Colony morphology Vegetative cell Akinete Heterocyte Reference 7 assignment 8 Sheath and shape Shape Width Length Shape Width min.- Length Shape Width Length 9 min.-max. min.-max. max. min.-max. min.-max. min.-max. (mean), (mean), (mean), (mean), (mean), (mean), 10 µm µm µm µm µm µm 11 12 Nostoc sp. PMC 881.14 France Epilithic nd nd 2.8 – 5.5 2.6 – 4.6 not nd nd +/- sub 2.6 – 5,7 2.3 – 5.1 This study biofilm (4.5) (3.4) observed spherical (3.4) (3.8) 13 Nostoc linckia IAM M-251 Japan nd nd nd nd nd nd nd nd nd nd nd Genbank site 14 web 15 Nostoc II Czech Field nd nd nd nd nd nd nd nd nd nd Rajaniemi et al muscorum Republic 2005 16 Nostoc V Czech Field nd nd nd nd nd nd nd nd nd nd Rajaniemi et al 17 ellipsosporum Republic 2005 18 Nostoc X Czech Field (salty Spherical or Barrel 3 - 4.2 nd not Mainly nd nd Singh et al 19 edaphicum Republic soils) oval sometimes shaped observed terminal, 2020 irregular rarely 20 colourless or intercalary 21 yellowish brown 22 Nostoc III Czech Field nd nd nd nd nd nd nd nd nd nd Rajaniemi et al calcicola Republic 2005 23 Nostoc sp. 1tu14s8 Finland Lake nd nd nd nd nd nd nd nd nd nd Rajaniemi et al 24 2005 25 Nostoc sp. ATCC 53789 Scotland thallus nd nd nd nd nd nd nd nd nd nd Genbank site web 26 Nostoc PCC 73102 Australia Root section, Dark blue green or Barrel 2.6 – 5.5 nd Present nd nd Barrel 4 -6.5 Singh et al 27 punctiforme Marozamia blackish spherical shaped to (3 - 5) shaped to (diameter) 2020 28 sp. (Humid sub aerophytic spherical to +/- sub 29 soil or cycad ellipsoidal spherical tubercules) 30 31 32 33 34 35 36 37 38 39 40 41 42 43 10 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Table S9. Comparison of the morphological features of the Aliinostoc sp. PMC 882.14 strain with other Aliinostoc and Trichormus species 4 belonging to the same phylogenetical cluster (Fig. 14). no: not observed. nd: not determined. 5 6 7 Filament Vegetative cell Akinete Heterocyte Reference 8 Taxonomic Strain Origin Habitat Sheath Shape of Shape Width Length Shape Width min.- Length Shape Width Length 9 assignment terminal min.-max. min.-max. max. min.-max. min.-max. min.-max. 10 cell (mean), (mean), (mean), (mean), (mean), (mean), 11 µm µm µm µm µm µm 12 Aliinostoc sp. PMC France Epilithic biofilm nd Conical Spherical 3.4 - 5.7 2.7 - 6.5 oval 4.6 - 7.4 4.5 - 9.1 Spherical, 4.1 - 6.5 4.2 - 6.9 (5.4) This study 13 882.14 /cylindrical (4.5) (4.6) (5.9) (7.2) intercalary (5.4) 14 Aliinostoc SA3O Iran Paddy field thin nd spherical 3.1 - 5.6 2.5 - 6.5 no nd nd spherical to 3.0 - 6.8 6.5 - 8.0 Kabirnataj et al. 15 constrictum hyaline / oblong 2020 cylindrical 16 17 Aliinostoc soli ZH1(3) India, Soil dwelling thin Bluish Barrel 3.2 – 3.7 3.6 – 4.0 Present nd nd spherical 3.7 – 4.9 3.8 – 4.3 Saraf et al. Pachmarh hyaline green shaped 2018 Kabirnataj 18 i mats with et al. 2020 19 leathery 20 texture 21 Aliinostoc LI PS India, Freshwater thin Greenish Barrel 5.2 - 5.6 5.3 – 5.6 Present nd nd spherical 3.7 – 4.9 3.8 – 4.3 Saraf et al. 22 tiwarii Mumbra hyaline blue mats shaped 2018 Kabirnataj 23 with soft et al. 2020 texture 24 25 Trichormus 1 nd nd nd Conical nd 1.6 - 3.0 lenticular 1.9 - 3.7 3.5 - 6.5 Rajaniemi et al dolium (2.3) (2.7) (4.6) 2005 26 27 Trichormus KINDAK Russia Soil nd Conical nd 2.1 -9.6 nd Oval, 5 – 7.4 8 – 14.8 nd nd nd Rajaniemi et al variabilis 2001/4 (6.7) slightly (6.9) (11.3) 2005 28 compress 29 ed in the 30 middle 31 Trichormus Kom-BAI nd nd nd Rounded nd 1.4 - 5.3 nd oval 7.5 - 7.5 12.5 - nd nd nd Rajaniemi et al 32 azollae (4) (7.5) 15 2005 33 (14.5) 34 Nostoc sp. PCC nd nd nd nd nd 3 - 3.5 no nd nd nd nd nd 35 7120 Anabaena BO Canada nd nd Conical nd 2.3 – 5.2 nd no nd nd nd nd nd Rajaniemi et al 36 oscillarioides HINDAK (3.8) 2005 37 1984/43 38 Minunostoc CHAB China Small seven- blue green Cylindrica nd 2.5 - 3.8 nd no nd nd no nd nd Cai et al. 2019 cylindricum 5843 hole Scenic gelatinous l (3.2) 39 area 40 41 42 43 11 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Table S10. Comparison of the morphological features of the Calothrix sp. PMC 884.14 strain with other Calothrix species belonging to the same 4 phylogenetical cluster (Fig. 14). nd: not determined. 5 6 Taxonomic Strain Origin Habitat Vegetative cell Akinete Heterocyte Reference 7 assignment 8 Shape Width min.-max. Length min.-max. Shape Shape Width min.- Length min.- 9 (mean), µm (mean), µm max. (mean), max. (mean), µm µm 10 11 12 13 Calothrix sp. PMC 884.14 France Epilithic nd 5.2 – 7.9 4.8 – 6.2 (5.5±0.4) not observed hemispherical 4.7-8.19 3.48-7.33 This study 14 biofilm (6.6±0.7) (6.2±0.9) (4.9±0.9) 15 Calothrix thermalis PCC 7715 France Thermal Isodiametric to nd nd Present nd nd nd PCC site web 16 spring longer than wide collection cells 17 18 Calothrix desertica PCC 7102 Chile Soil, fine nd nd nd nd nd nd nd PCC site web desert sand collection 19 20 Calothrix sp. PCC 7103 USA Herbarium nd nd nd nd nd nd nd PCC site web material of collection 21 Anacystis 22 montana 23 Calothrix parietina CCAP 1410/10 England Freshwater nd nd nd nd nd nd nd Genbank site 24 web 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 12 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 Table S11. Molecules selected for their antioxidant and/or anti-inflammatory properties (Demay et al. 4 2019) related to the genera of cyanobacteria isolated from Thermes de Balaruc-Les-Bains. MAAs: 5 Mycosporine-like amino acids. 6 7 Targeted molecule Characteristics Bioactivities Genus producers References 8 families 9 No cytotoxicity 10 Peptide Anti-inflammatory Linear activity 11 Aeruginosins Nostoc sp. Lukešová 30/93 [1–4] 12 NRPS Protease inhibitor 13 Hydrosoluble (trypsin, thrombin, plasmin) 14 15 Terpenoid Antioxidant 16 Carotenoids Pigment All cyanobacteria [5–10] Sunscreen 17 Liposoluble 18 Antioxidant Substituted tetra- 19 Antimutagenic pyrrole porphyrin 20 Chemopreventive Chlorophylls molecule All cyanobacteria [11–13] 21 Photosensitizing act. Pigment Cancer preventive 22 Liposoluble 23 agent 24 Anti-inflammatory Lactone activity Leptolyngbya crossbyana 25 Honaucins Linear No antioxydant act. [14,15] HI09-1 26 Liposoluble Quorum sensing 27 inhibition 28 Calothrix parietina 29 Calothrix sp. 30 Leptolyngbya sp. 31 Lyngbya aestuarii Lyngbya sp. CU2555 Antioxidant 32 Lyngbya sp. Cyclohexenone- UV-absorbing 33 Mycosporine-like Microcoleus chthonoplastes amino acid UV-protective act. [5,16–25] 34 amino acids (MAAs) Microcoleus paludosus Hydrosoluble Sunscreen Microcoleus sp. 35 Antiphotoaging 36 Nostoc commune 37 Nostoc microscopicum Nostoc punctiforme ATCC 38 29133 39 Nostoc sp. 40 Antioxidant 41 Anti-inflammatory Protein Neuroprotective 42 Phycocyanins Pigment All cyanobacteria [26–29] effects 43 Hydrosoluble Hepatoprotective 44 effects 45 Protein Antioxidant Several strains belonging to all 46 Phycoerythrins Pigment [30–32] Moderate aging orders 47 Hydrosoluble 48 Calothrix crustacea 49 Calothrix parietina Calothrix sp. 50 Anti-inflammatory Chroococcus sp. 51 UV protection Lyngbya aestuarii Inhibit cell cycle 52 Alkaloid Lyngbya sp. Scytonemins kinases 53 Pigment Lyngbya sp. CU2555 [16,33–38] Antiproliferative 54 Liposoluble Nostoc commune Vauch Induction of Nostoc microsopicum 55 autophagic death 56 Nostoc parmelioides 57 Nostoc pruniforme Nostoc punctiforme PCC 58 73102, ATCC 29133 59 60

13 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 References Table S11 4 5 6 1. Ishida, K.; et al. Aeruginosins, protease inhibitors from the cyanobacterium Microcystis 7 aeruginosa. Tetrahedron 1999, 55, 10971–10988, doi:10.1016/S0040-4020(99)00621-3. 8 2. Matsuda, H.; et al. Aeruginosins 102-A and B, New Thrombin Inhibitors from the Cyanobacterium 9 Microcystis viridis (NIES-102). Tetrahedron 1996, 96. 10 3. Fewer, D.P.; et al. New Structural Variants of Aeruginosin Produced by the Toxic Bloom Forming 11 Cyanobacterium Nodularia spumigena. PLoS One 2013, 8, doi:10.1371/journal.pone.0073618. 12 4. Kapuścik, A.; et al. Novel aeruginosin-865 from Nostoc sp. as a potent anti-inflammatory agent. 13 14 ChemBioChem 2013, 14, 2329–2337, doi:10.1002/cbic.201300246. 15 5. Choi, H.; et al. Honaucins A-C, potent inhibitors of inflammation and bacterial quorum sensing: 16 Synthetic derivatives and structure-activity relationships. Chem. Biol. 2012, 19, 589–598, 17 doi:10.1016/j.chembiol.2012.03.014. 18 6. Mascuch, S.J.; et al. Marine Natural Product Honaucin A Attenuates Inflammation by Activating 19 the Nrf2-ARE Pathway. J. Nat. Prod. 2017, 81, 506–514, doi:10.1021/acs.jnatprod.7b00734. 20 7. Rastogi, R.P.; et al. Characterization of UV-screening compounds, mycosporine-like amino acids, 21 22 and scytonemin in the cyanobacterium Lyngbya sp. CU2555. FEMS Microbiol. Ecol. 2014, 87, 23 244–256, doi:10.1111/1574-6941.12220. 24 8. Garcia-Pichel, F.; et al. Occurrence of UV-Absorbing, Mycosporine-Like Compounds among 25 Cyanobacterial Isolates and an Estimate of Their Screening Capacity. Appl. Environ. Microbiol. 26 1993, 59, 163–9. 27 9. Shibata, K. Pigments and a UV-absorbing substance in corals and a blue-green alga living in the 28 great barrier reef. Plant Cell Physiol. 1969, 10, 325–335, doi:10.1093/oxfordjournals.pcp.a074411. 29 30 10. Jain, S.; et al. Cyanobacteria as efficient producers of mycosporine-like amino acids. J. Basic 31 Microbiol. 2017, 57, 715–727. 32 11. Kageyama, H.; et al. Mycosporine-Like Amino Acids as Multifunctional Secondary Metabolites in 33 Cyanobacteria: From Biochemical to Application Aspects; 1st ed.; Elsevier B.V., 2018; Vol. 59; 34 ISBN 9780444641793. 35 12. Ryu, J.; et al. Protective effect of porphyra-334 on UVA-induced photoaging in human skin 36 37 fibroblasts. Int. J. Mol. Med. 2014, 34, 796–803, doi:10.3892/ijmm.2014.1815. 38 13. Suh, H.-J.; et al. Mycosporine Glycine Protects Biological Systems Against Photodynamic 39 Damage by Quenching Singlet Oxygen with a High Efficiency¶. Photochem. Photobiol. 2003, 78, 40 109, doi:10.1562/0031-8655(2003)078<0109:mgpbsa>2.0.co;2. 41 14. Matsui, K.; et al. Novel glycosylated mycosporine-like amino acids with radical scavenging 42 activity from the cyanobacterium Nostoc commune. J. Photochem. Photobiol. B Biol. 2011, 105, 43 81–89, doi:10.1016/j.jphotobiol.2011.07.003. 44 45 15. Sakamoto, T.; et al. Four chemotypes of the terrestrial cyanobacterium Nostoc commune 46 characterized by differences in the mycosporine-like amino acids. Phycol. Res. 2019, 67, 3–11, 47 doi:10.1111/pre.12333. 48 16. Karsten, U.; et al. Carotenoids and mycosporine-like amino acid compounds in members of the 49 Genus microcoleus (Cyanobacteria): A chemosystematic study. Syst. Appl. Microbiol. 1996, 19, 50 285–294, doi:10.1016/S0723-2020(96)80054-3. 51 17. Sinha, R.P.; et al. Database on mycosporines and mycosporine-like amino acids (MAAs) in fungi, 52 53 cyanobacteria, macroalgae, and animals. J. Photochem. Photobiol. B Biol. 2007, 89, 54 29–35, doi:10.1016/j.jphotobiol.2007.07.006. 55 18. Stevenson, C.S.; et al. Scytonemin-a marine natural product inhibitor of kinases key in 56 hyperproliferative inflammatory diseases. Inflamm. res 2002, 51, 112–114. 57 19. Stevenson, C.S.; et al. The identification and characterization of the marine natural product 58 scytonemin as a novel antiproliferative pharmacophore. J. Pharmacol. Exp. Ther. 2002, 303, 858– 59 60 866, doi:10.1124/jpet.102.036350.ever.

14 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.12.422513; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 3 20. Itoh, T.; et al. Reduced scytonemin isolated from Nostoc commune induces autophagic cell death 4 in human T-lymphoid cell line Jurkat cells. Food Chem. Toxicol. 2013, 60, 76–82, 5 6 doi:10.1016/j.fct.2013.07.016. 7 21. Pathak, J.; et al. Genetic regulation of scytonemin and mycosporine-like amino acids (MAAs) 8 biosynthesis in cyanobacteria. Plant Gene 2019, 17, 100172, doi:10.1016/J.PLGENE.2019.100172. 9 22. Fleming, E.D.; et al. Effects of nitrogen source on the synthesis of the UV-screening compound, 10 scytonemin, in the cyanobacterium Nostoc punctiforme PCC 73102. FEMS Microbiol. Ecol. 2008, 11 63, 301–308, doi:10.1111/j.1574-6941.2007.00432.x. 12 23. Garcia-Pichel, F.; et al. Characterization and biological implications of scytonemin, a 13 14 cyanobacterial sheath pigment. J. Phycol. 1991, 27, 395–409, doi:10.1111/j.0022- 15 3646.1991.00395.x. 16 24. Stahl, W.; et al. Antioxidant activity of carotenoids. Mol. Aspects Med. 2003, 24, 345–351. 17 25. Stahl, W.; et al. Bioactivity and protective effects of natural carotenoids. Biochim. Biophys. Acta - 18 Mol. Basis Dis. 2005, 1740, 101–107, doi:10.1016/j.bbadis.2004.12.006. 19 26. Hirschberg, J.; et al. Carotenoids in Cyanobacteria BT - The Molecular Biology of 20 Cyanobacteria. In The Molecular Biology of Cyanobacteria; Springer Netherlands: Dordrecht, 21 22 1994; pp. 559–579 ISBN 978-94-011-0227-8. 23 27. Joshi, D.; et al. Effect of UV-B Radiation and Desiccation Stress on Photoprotective Compounds 24 Accumulation in Marine Leptolyngbya sp. Appl. Biochem. Biotechnol. 2018, 184, 35–47, 25 doi:10.1007/s12010-017-2523-3. 26 28. Takaichi, S.; et al. Unique carotenoids in the terrestrial cyanobacterium nostoc commune nies-24: 27 2-hydroxymyxol 2??-fucoside, nostoxanthin and canthaxanthin. Curr. Microbiol. 2009, 59, 413– 28 419, doi:10.1007/s00284-009-9453-4. 29 30 29. Lanfer-Marquez, U.M.; et al. Antioxidant activity of chlorophylls and their derivatives. In 31 Proceedings of the Food Research International; Elsevier, 2005; Vol. 38, pp. 885–891. 32 30. Queiroz Zepka, L.; et al. Catabolism and bioactive properties of chlorophylls. Curr. Opin. Food 33 Sci. 2019, 26, 94–100, doi:10.1016/j.cofs.2019.04.004. 34 31. Ferruzzi, M.G.; et al. Digestion, absorption, and cancer preventative activity of dietary 35 chlorophyll derivatives. Nutr. Res. 2007, 27, 1–12. 36 37 32. Romay, C.; et al. C-Phycocyanin: A Biliprotein with Antioxidant, Anti-Inflammatory and 38 Neuroprotective Effects. Curr. Protein Pept. Sci. 2003, 4, 207–216, 39 doi:10.2174/1389203033487216. 40 33. Remirez, D.; et al. Influence of C-phycocyanin on hepatocellular parameters related to liver 41 oxidative stress and Kupffer cell functioning. Inflamm. Res. 2002, 51, 351–356, 42 doi:10.1007/PL00000314. 43 34. Patel, A.; et al. Antioxidant potential of C-phycocyanin isolated from cyanobacterial species 44 45 Lyngbya, Phormidium and Spirulina spp. Indian J. Biochem. Biophys. 2006, 43, 25–31, 46 doi:10.1016/S0260-8774(98)00168-X. 47 35. Romay, C.; et al. Antioxidant and anti-inflammatory properties of C-phycocyanin from blue- 48 green algae. Inflamm. Res. 1998, 47, 36–41, doi:10.1007/s000110050256. 49 36. Sonani, R.R.; et al. Concurrent purification and antioxidant activity of phycobiliproteins from 50 Lyngbya sp. A09DM: An antioxidant and anti-aging potential of phycoerythrin in Caenorhabditis 51 elegans. Process Biochem. 2014, 49, 1757–1766, doi:10.1016/j.procbio.2014.06.022. 52 53 37. Patel, S.N.; et al. Antioxidant activity and associated structural attributes of Halomicronema 54 phycoerythrin. Int. J. Biol. Macromol. 2018, 111, 359–369, doi:10.1016/j.ijbiomac.2017.12.170. 55 38. Bryant, D.A. Phycoerythrocyanin and phycoerythrin: properties and occurrence in cyanobacteria. 56 J. Gen. Microbiol. 1982, 128, 835–844, doi:10.1099/00221287-128-4-835. 57 58 59 60

15