Specimens of C. surera Parodi et Cáceres subsp. nov. were collected in Las Cascadas, Necochea, Buenos Aires Province (38°27′39′′ S 58°45′39′′ W) in April 2015 (Supplementary Figure S1). The water salinity at this site was < 1‰, measured with a Salinity Refractometer S/MIII, Cat No. 2441, ATAGO CO., LTD. Sulfate content of water at the collection site was determined by ion exchange chromatography with conductimetric detection using a DIONEX DX-100 chromatography system (Sunnyvale, CA, United States) with an AS4A column (4mm × 250 mm), an AMMS-II micromembrane suppressor; elution was carried out with 1.8 mM Na₂CO₃/1.7 mM NaHCO₃, at a flow rate of 2 mL min^-1.

Cladophora surera grew free floating in the freshwater course. Sporophytic and gametophytic plants are isomorphic. Measurements of length and width of cells from the principal axis and branches were within the range reported for this species by Parodi and Cáceres (1995). Gametangia developed from the upper cells in ultimate ramifications with similar dimensions and appearance to the vegetative cells. The samples used in this work were in the vegetative state. Thalli of the algae were washed with distilled water and analyzed for epiphytic and epizoic contaminants in a Nikon AFX-II macroscope (Nikon, Japan).

Genomic DNA was obtained from fresh algal material of C. surera using REDExtracts-N-Amp Tissue PCR Kit (SIGMA-ALDRICH). The nuclear-encoded small subunit (LSU) rDNA gene fragment was amplified and sequenced using primers and temperature profile as indicated in Boedeker and Immers (2009). Sequences were inspected and aligned using Geneious version 7.0 (Biomatters). SSU sequence was deposited in Gene Bank under accession number MF001434. The position of C. surera was investigated through the phylogenetic analyses of Thirty-two species of Cladophorales (Ulvophyceae), representing marine, brackish and freshwater environments (Accession numbers in Figure 1B). Two species from Ulvophyceae (U. curvata and Ulothrix zonata) were included as outgroups. Phylogenetic analyses of molecular characters were performed through Bayesian analysis (BA) as implemented in BEAST v1.6.2 (Drummond and Rambaut, 2007; Heled and Drummond, 2010). The model of sequence evolution was GTR+I+G (Rodríguez et al., 1990; Yang, 1994; Gu et al., 1995). Calibration of molecular clock was performed using the estimated minimum age of 600–570 million years for Cladophorales (van den Hoek and Chihara, 2000; Leliaert et al., 2007) based on fossil data. An uncorrelated relaxed clock model and a log-normal fossil calibration was used during tree searches. The number of Markov Chain Monte Carlo (MCMC) iterations was 30000000, from which the first 3000000 were discarded as non-converged burn-in; nodal support values were given as posterior probabilities.

For light microscopy (LM) semithin sections (10 μm) were mounted on glass slides and then observed with a Carl Zeiss Axiolab microscope (Carl Zeiss, Jena, Germany). The staining procedures used in LM histochemical characterization based on Krishnamurthy (1999), were carried out on the fixed tissues described above and included Toluidine Blue O (TBO; 0.05% w/v) in 0.1 m HCl at pH 1.0 that stains sulfated polysaccharides (red–purple, c metachromasia), and TBO at pH7 (for negatively charged polysaccharides). Hot water extraction at 90°C was carried out on cross-sections, which were then stained in conditions described above.

Algal samples of C. surera were dried in open air. The milled algae (100 g) were extracted twice with EtOH 70% (20g/L) for 3 h at room temperature. The residue from the alcohol extraction was extracted for 3 h with H₂O (20 g/L) at 90°C, giving extract Cs90 (20.1 g). Cs90 was dissolved in water (4.5 mg/ml). A residue (Ri) was separated from the supernatant, which was chromatographed on DEAE-Sephadex A-25. The supernatant was applied to a column (90 cm × 1.5 cm id), previously stabilized in H₂O. The first elution solvent was water and then NaCl solutions of increasing concentration up to 4 M. Fractions of 4 ml were collected. Finally, the phase was boiled in 4 M NaCl solution. The presence of carbohydrates in the samples was detected by the phenol sulfuric acid method (Dubois et al., 1956); after obtaining blank readings, the eluant was replaced by another with higher concentration of NaCl. Seven fractions (F1-F7) were obtained, dialyzed (molecular weight cut off 3,500) and freeze dried (Supplementary Figure S2).

The total sugars content was analyzed by the phenol-sulfuric acid method (Dubois et al., 1956). Sulfate was determined turbidimetrically (Dodgson and Price, 1962). Alternatively, ion exchange chromatography with conductimetric detection was used: the sample was hydrolyzed in 2 M CF₃COOH at 121°C for 2 h, evaporated to dryness under nitrogen and redissolved in high purity water from a Milli-Q system. A DIONEX DX-100 chromatography system (Sunnyvale, CA, United States) was used with an AS4A column (4 × 250 mm), an AMMS-II micromembrane suppressor and a conductivity detector, elution was carried out with 1.8 mM Na₂CO₃/1.7 mM NaHCO₃, at a flow rate of 2 mL min^-1. The absence of pyruvic acid and uronic acids was confirmed using the colorimetric determinations of Koepsell and Sharpe (1952) and Filisetti-Cozzi and Carpita (1991). The protein content was measured by microanalysis to determine the amount of nitrogen, a factor of 5 was applied to calculate the amount of protein, according to Angell et al. (2016). The configuration of galactose and arabinose was determined by the method of Cases et al. (1995) through their diastereomeric acetylated 1-deoxy-1-(2-hydroxypropylamino) alditols. To determine the monosaccharide composition, samples were derivatized to the alditol acetates (Stevenson and Furneaux, 1991).

The sample (10–20 mg) was converted into the corresponding triethylammonium salt (Stevenson and Furneaux, 1991) and methylated according to Ciucanu and Kerek (1984). The sample was dissolved in dimethylsulfoxide; finely powdered NaOH was used as base. The methylated samples were submitted to reductive hydrolysis and acetylation to give the alditol acetates in the same way as the parent polysaccharides (Stevenson and Furneaux, 1991).

GC of the alditol acetates were carried out on a Agilent 7890A gas-liquid chromatograph (Avondale, PA, United States) equipped with a flame ionization detector and fitted with a fused silica column (0.25mm i.d. × 30 m) WCOT-coated with a 0.20 mm film of SP-2330 (Supelco, Bellefonte, PA, United States). Chromatography was performed: from 200 to 240°C at 2°C min^-1, followed by a 10-min hold for alditol acetates. For the partially methylated alditol acetates, the initial temperature was 160°C, which was increased at 1°C min^-1 to 210°C and then at 2°C min^-1 to 230°C. N₂ was used as the carrier gas at a flow rate of 1 mL min^-1 and the split ratio was 80:1. The injector and detector temperature was 250°C.

GC–MS of the partially methylated alditol acetates was performed on a Agilent 7890A gas-liquid chromatograph equipped the SP-2330 interfaced to a Agilent 5977A Series mass spectrometer, working at 70 eV. The flow rate was 1.3 ml min^-1, the injector temperature was 250°C. Mass spectra were recorded over a mass range of 30–500 amu.

The reaction was carried out by the microwave-assisted method described by Navarro et al. (2007). The sample (40 mg) was converted to the pyridinium salt and dissolved in 10 ml of DMSO containing 2% of pyridine. The mixture was heated for 10 s intervals and cooled to 50°C (× 6). It was dyalized 3 days against tap water and then 24 h against distilled water (MWCO 3,500) and lyofilized. An aliquot was methylated as described above without previous isolation of the product.

The reaction was carried out according to Bilan et al. (2007). The sample (100 mg) was heated in 1% CH₃COOH (20 mL) for 4 h at 100°C, the solution was neutralized with NaHCO₃, dialyzed, and lyophilized to give RiH (78.2 mg). RiH was fractionated by anion exchange chromatography on DEAE-Sephadex A-25 in a similar way as F1, in this case, five fractions were obtained (RiH-F1-RiH-F5).

Samples were analyzed on a Thermo Scientific Nicolet 6700 spectrometer equipped with a smart ARK (Attenuated Reflectance Kit) accessory using a standard ZnSe crystal and a DTGS KBr detector. The spectra were recorded in the 650–4000 cm^-1 range, and the spectral resolution was 4 cm^-1. Data were processed by using the software Origin Pro 9.0.0.

500 MHz¹H NMR, proton decoupled 125 MHz ¹³C NMR spectra, and two-dimensional NMR experiments (HSQC, HMBC, and COZY) were recorded on a Bruker AM500 at room temperature, with external reference of TMS. The samples (20 mg) were exchanged in 99.9% D2O (0.5 mL) four times. Chemical shifts were referenced to internal acetone (δ_H 2.175, δ_CH3 31.1). Parameters for ¹³C NMR spectra were as follows: pulse angle 51.4°, acquisition time 0.56 s, relaxation delay 0.6 s, spectral width 29.4 kHz, and scans 25,000. For ¹H NMR spectra the parameters were: pulse angle 76°, acquisition time 3 s, relaxation delay 3 s, spectral width 6250 Hz and scans 32. 2D spectra were obtained using standard Bruker software.

Specimens of the green alga were collected in a freshwater environment (Quequén Grande river, 38°27′39′′ S 58°45′39′′ W) located in Buenos Aires Province, Argentina (Supplementary Figure S1). Based on the morphological characters, it was assigned to C. surera (Figure 1A; Parodi and Cáceres, 1991, 1995).

In order to gain further insight on the taxonomical identity, a molecular characterization of the SSU (nuclear-encoded small subunit rDNA) gene fragment of the collected species was carried out and its phylogenetic position within the Cladophorales was investigated. The Bayesian tree obtained (Figure 1B) recovered with high Posterior Probabilities (PP) the three major groups previously described by Boedeker et al. (2012): the “Aegagropila clade,” the “Siphonocladus clade” and the “Cladophora clade.” C. surera is resolved within the latter group, and particularly within a lineage of four species that can be mainly found in freshwater/brackish environments (Hanyuda et al., 2002; Boedeker et al., 2012). Within the order Cladophorales, the boundary between marine and freshwater environments was crossed at least two to three times (van den Hoek, 1963; Logares et al., 2007; Hayakawa et al., 2012; Boedeker et al., 2016; Figure 1B). Calibration of molecular clock with fossil data suggests that colonization of freshwater environments probably occurred 11.4 million years ago (MYA; 4–25 MYA 95% PP) by the ancestor of the four freshwater species closely related to C. surera (Figure 1B); or even before, at about 65.5 MYA (35–115 MYA 95%PP), during the diversification of the more basal species Rhizoclonium hieroglyphicum.

Cladophora surera showed a thick cell wall with two fibrillar-like layers delimiting a middle amorphous region. TBO staining suggested the presence of sulfated polysaccharides concentrated in both marginal cell wall layers (Figure 1C). Hot water extraction (at 90°C) carried out on cross-sections greatly suppressed TBO staining (Figure 1D). In agreement, the hot water extract (Cs90) obtained from C. surera that represents 20.1% (w/w) of the algal dry weight (Supplementary Figure S2) contained high amounts of sulfate (17%, as SO₃Na, which correspond to ∼4% w/w of the algal dry weight), in accordance with the histochemical stainings.

Finally, Attenuated Total Reflection-Fourier Transformed Infra-Red (ATR-FTIR) spectrum of the dry milled alga, as well as that of extract Cs90 (Figure 1E) showed the bands diagnostic for the presence of sulfate ester groups at 1230 cm^-1, which was assigned to asymmetric stretching of O = S = O, and at 825 cm^-1, due to stretching of C_ecuatorial-O-S and/or C_primary-O-S (Prado Fernández et al., 2003). In agreement, after exhaustive sequential extraction with water (at 90°C) and with alkaline solutions, both IR bands (825 and 1230 cm^-1) mostly disappeared in the remaining cell wall residue (FR) confirming that sulfated polysaccharides are confined in the aqueous extracts (Figure 1E).

All these results together confirmed the presence of high quantities of sulfated polysaccharides in cell walls of freshwater macroalga C. surera. The water salinity at the collection site was < 1‰ (w/w; as NaCl) and the sulfate content of the water was ∼1 mM, while usually in marine waters there are high sulfate concentrations (∼25–28 mM) (Bochenek et al., 2013). These results suggested that sulfated polysaccharides in some Cladophora species (at least in C. glomerata, and now in C. surera) could be synthetized in response to other stress factors, like desiccation and changes in ionic strength of the environment, not linked to high salinity levels as a stable environmental condition.

In order to characterize in depth the structure of these sulfated polysaccharides, with emphasis in the position of the sulfate groups on the carbohydrates backbone, the monosaccharide composition of the main cell wall extract obtained with hot water (Cs90) was determined. Cs90 is composed by arabinose and galactose as major monosaccharides, also important amounts of xylose were present (Table 1). Methylation analysis of Cs90 showed major quantities of 2,3-di-O-methyl-, 2-O-methyl-, 3-O-methyl-, and non-methylated arabinose, which correspond to 4-linked non-substituted, 3-substituted, 2-substituted, and disubstituted arabinose units (17, 28, 20, and 28 per 100 arabinose units, respectively) in the polysaccharide backbone. Desulfation of Cs90 gave Cs90D (Table 1), which by methylation analysis showed an important increase in 4-linked non-substituted arabinose units (A, ∼50% of the total arabinose units), and a decrease of 3-substituted (A3S) and 2,3-disubstituted units (A2,3S), while the percentage of 4-linked 2-substituted (A2X + A2R, R = chains up to four residues of Galp) units also increased to 38%. These results indicate that sulfate groups were mainly linked to C3 of the arabinopyranose units and in minor amounts, to C2 and C3, while some arabinose units were sulfated on C3 and substituted with single units of xylose or galactose on C2. Only minor amounts of galactofuranose units were detected. These units were previously found in important quantities in the arabinan of C. falklandica (Arata et al., 2016), and they were also detected in C. rupestris (Percival and McDowell, 1981, and references therein). Besides, methylation and desulfation–methylation analysis suggested the presence of short side chains of 3- and 3,6-linked galactopyranose units, partially sulfated on C4.

As Cs90 had a limited solubility in water, it was not possible to carry out NMR spectroscopic analysis of this extract, however, good spectra were obtained from Cs90D (Figure 2A and Table 2), which allowed to determine that all the major monosaccharide units described above were in the β-configuration, and to confirm the linkages between them.

Besides, a strategy comprising controlled acid hydrolysis, fractionation by anion exchange chromatography (Supplementary Figure S2), and structural elucidation of the fractions by methylation analysis and NMR spectroscopy (Figures 2A,B and Table 2) was performed. Similar structural features were found in all the fractions obtained, but in different quantities. The highly purified sample FiH3 is shown as example (Figure 2B, and Tables 1, 2), and confirmed the structural features of these polysaccharides. As expected for FiH3, a fraction which eluted from an anion exchange chromatographic column with 2 M NaCl (Supplementary Figure S2), it is highly sulfated, and disulfated arabinose units are present in important amounts. In addition, it was found that in this fraction the β-D-galactopyranose units were mostly 6-linked and sulfated on C3. This structure was proposed based on methylation analysis, which showed the presence of 2,4-di-O-methylgalactose, as major partially methylated galactose derivative, and by the absence of a signal at δ 83.0/3.80 which would correspond to 3-linked β-D-galactopyranose units. Moreover, a small signal at δ 81.0/4.28 is a strong evidence for the presence of the mentioned 6-linked 3-sulfated galactose units. This substitution pattern of the galactan moiety was similar to that found in marine C. socialis (Sri Ramana and Venkata Rao, 1991). On the other hand, in C. falklandica, most of the galactose was in the furanosic form, and only minor quantities of 3-linked mostly 6-sulfated galactopyranose units were detected. Here, the important amount of terminal galactopyranose units, detected by methylation analysis and confirmed in the NMR spectra of Cs90D (Figure 2A and Table 2), indicates possible small side chains of up to ∼4 units of the arabinan backbone. These results suggest that the most important structural differences between the sulfated polysaccharides from species of Cladophora studied so far are restricted to the galactose units, while the pyranosic arabinan backbone and its sulfation pattern is conserved.

In conclusion, sulfated xylogalactoarabinans from cell walls of the freshwater alga C. surera have structural features similar to those reported for the marine species of this genus. Based on the structural determination carried out by linkage analysis and NMR spectroscopy, it is proposed that C. surera biosynthesizes sulfated xylogalactoarabinans as shown in Figure 2C.

The presence of sulfated polysaccharides in C. surera is in agreement with two recent studies on C. glomerata from different freshwater environments (Nan river in Thailand and Lake Oporzynskie in Poland) (Pankiewicz et al., 2016; Surayot et al., 2016). In all three cases, they developed in freshwater environments. Sulfated polysaccharides are widespread in the cell walls of marine angiosperms, in marine algae (green, red, and brown seaweeds), in the extracellular matrix of vertebrate tissues, and in invertebrate species (Aquino et al., 2005; Ghandi and Mancera, 2008; Pomin and Mourao, 2008; Usov and Bilan, 2009; Ciancia et al., 2010; Usov, 2011). In the green alga Lamprothamnium papulosum (Characeae, Charophyta), which grows in water of fluctuating salinity, the extracellular sulfated mucilage increases in sulfate content with increasing salinity (Shepherd and Beilby, 1999; Shepherd et al., 1999), while in brown algae the concentrations of sulfate groups on fucans positively correlate with increasing exposure to the atmosphere in the intertidal zone, suggesting a role in desiccation resistance (Mabeau and Kloareg, 1987). Ectocarpus subulatus (Phaeophyta) isolated from a true freshwater environment (West and Kraft, 1996) undergoes major morphological, transcriptomic and metabolic changes under variable salinities, including alteration of the expression of genes encoding enzymes potentially involved in the sulfation or de-sulfation of cell wall polysaccharides (Dittami et al., 2012). It is now widely accepted that the occurrence of sulfated polysaccharides in phylogenetically distant taxa is a case of convergent adaptation to a broad range of environmental conditions, such as stable high ionic media (e.g., seawater for marine plants and algae) or transient changes in salt concentration linked to desiccation and high temperature stresses (e.g., in some cases of freshwater macroalgae and vascular plants) (Mabeau and Kloareg, 1987; Shepherd and Beilby, 1999; Shepherd et al., 1999; Aquino et al., 2011; Dantas-Santos et al., 2012; Pankiewicz et al., 2016; Surayot et al., 2016). From a physiological point of view, sulfated cell wall polysaccharides from marine plants and marine algae may confer an adaptive advantage through possible structural and osmotic functions that are linked to an environmental pressure. Terrestrialization, and the subsequent taxonomic proliferation of land plants, was predated by a divergence between the Chlorophyte (a group of green algae) and the Streptophyte lineages (which includes land plants). This is strongly correlated with a change in habitat preference from saline to freshwater conditions with low or no salt, as well as exposure to transient changes in ionic strength (e.g., caused by water evaporation in small microsites). These transitions are accompanied by a major alteration in cell wall polysaccharides composition such that, although the majority of marine Chlorophytes contain sulfated cell wall polysaccharides, they are largely absent from most of the freshwater Charophycean green algae and their descendants. The latter green algae contain large amounts of negatively charged pectins with carboxylated sugars (uronic acids) in their cell walls that could functionally replace some of the roles of sulfated polysaccharides (Domozych et al., 2012). Further studies to determine if sulfated polysaccharides are present in other freshwater algae are required to obtain a more comprehensive picture about their possible functions in the cell wall.