Water at the sampling site was pH = 7.1 and nearly warm (23 °C) at the sampling time. Nitrate (mg L-1) and phosphate (mg L-1) concentrations were 0.09 and 0.1, respectively. Dissolved oxygen (ppm) and conductivity (mS/cm) were 12.1±1.61 and 905.8±3.25, respectively.
Morphology and taxonomic identification
A morphological comparison between isolated (D. alborizicum) and the other species of Demonostoc including type of the genus, represented by the reference strain D. muscorum NIVA-CYA 818 , is presented in Table 1.
Macroscopically, isolate 1387 grew attached to the wall of a qanat, exhibiting dark green color. Laboratory observations revealed that it was able to grow fast as a gelatinous biomass, dull olive green, forming a wide range of macroscopic colonies on agar plates (Fig. 1, a). Further, cells colour changed from light green to dark green with the growth progression in plates.
Other morphological characteristics were observed as filaments were generally straight, but sometimes could be twisted, bet or sigmoid (Fig. 1, b). Each filament was a thin, colorless, hyaline sheath (Fig. 1, c). The trichomes were very thin and strongly constricted and the cells were spherical until ellipsoidal that could be much longer than wide (Fig. 1, a-c).
Also were presented about spherical to oblong terminal and oblong intercalary heterocytes, sometimes the terminal heterocytes formed two or three in chain (Fig. 1, d). The reproduction was for fragmentation and formed hormogonia (Fig. 1, e).
The akinetes as well were about terminal and intercalary and formed single or in chain (Fig. 1, f).
The morphology of isolate 1387 is consistent with Desmonostoc, because its present typical characteristics of the genus, as cells longer than wide and akinetes that form in chains.
The strain 1387 is morphologically distinct from other species of Demonostoc in the present of filaments generally straight, thinner trichomes and largest length-width ratio (Table 1), which means that it has the longest cells of all the species of the genus with respect to the width of the trichome. These characteristics could indicate that the strain is a new species.
Life cycle of strain 1387
Trichomes at the onset of incubation, variously-sized Hormogonia (Fig.2, a) tended to prevail (2 to 10 cells). Hormogonial cells were elongated as well as the cells in vegetative filaments, with tapered terminal cells. These characteristic terminal cells eventually differentiate into heterocytes as the filaments return to the vegetative growth state (Fig.2, b, c). After a short growth period (1–2 days), the formation of pre-heterocytes and heterocytes (Fig. 2, d) intercalary took place in long filaments (15 to 35 cells). Vegetative cells are spherical or slightly oblong, in young filaments, with progress of growth, they became oblong and ellipsoidal (Fig.2, a, d). Degenerated vegetative cells, like necridic cells, also appeared as empty cells, which eventually disintegrated or became detached, resulting in filament fragmentation, detached heterocytes as single or attached together as a group were also observed (Fig. 2, e). In some cases, the space of heterocytes and vegetative cells became longer than usual (Fig. 2, b, d, e). Akinetes were found as single and separated from the main filaments, or as a chain at the end or in the middle of filaments adjacent to heterocytes (Fig. 1, f; Fig. 2, f).
A 16S rDNA fragment was sequenced and aligned with other 166 nucleotide sequences of cyanobacteria obtained from GenBank for phylogenetic analysis. The maximum Likelihood (ML) phylogenetic analysis is shown in Fig. 3.
Although the 16S rRNA gene sequence coupled with its 16S–23S rRNA internal transcribed spacer (ITS) secondary structures represent the core of the present manuscript, the mcyG and mcyD gene sequences were used as additional molecular markers for characterization of D. alborizicum. Different genera and families were used in the construction of the phylogenetic tree, as shown in the figure, each genus is placed in a separate cloud with other similar genera. In addition, the genus Desmonostoc has been placed in a separate cloud together with Nostoc_muscorum_Ind33, Nostoc_sp_PCC_7906, Desmonostoc_punense_MCC_2741, Nostoc_muscorum_UTAD_N213, Desmonostoc_sp_PCC_7422, Desmonostoc_sp_SA25, Desmonostoc_magnisporum_AR6_PS and Desmonostoc_alborizicum. In fact, the studied strain with the strain Desmonostoc_magnisporum AR6 PS (MH497066) is in the same branch with phylogenetic similarity 86.5 percent.
A 1263 and 494 bp nucleotide sequence has successfully been amplified and sequenced for the mcyD and mcyG genes using specific primers (Table S3). The highest mcyD and mcyG sequences similarity was found to be 100% and 91.9% of identity with Nostoc_sp__IO-102-I (AY566857) and Nostoc_sp__CENA88 (GQ259210) respectively (Fig. 4). All representatives of mcyG and mcyD gene sequences are grouped together in the same clade in the phylogenetic tree. To date, there is no record of mcyD and mcyG sequences from Desmonostoc in the database.
According to the phylogenetic tree we have also compared the 16S rRNA and ITS p-distances of our strain with related genera namely (Nostoc_muscorum_Ind33, Nostoc_sp_PCC_7906, Desmonostoc_punense_MCC_2741, Nostoc_muscorum_UTAD_N213, Desmonostoc_sp_PCC_7422, Desmonostoc_sp_SA25, Desmonostoc_magnisporum_AR6_PS and Desmonostoc_alborizicum) and (Desmonostoc_salinum_CCM-UFV059, Desmonostoc_alborizicum, Desmonostoc_sp._111_CR4_BG11B, Desmonostoc_magnisporum_AR6_PS, Desmonostoc_sp._CCIBT_3489, Desmonostoc_sp._111_CR4_BG11N and Desmonostoc_sp._CCIBt3489_clone_53) respectively.
Results showed that Desmonostoc_alborizicum shared a 16S rRNA sequence similarity of 97.27% with Nostoc_muscorum_Ind33, 97.84% with Nostoc_sp_PCC_7906, 97.7% with Desmonostoc_punense_MCC_2741, 97.85% with Nostoc_muscorum_UTAD_N213, 97.99% with Desmonostoc_sp_PCC_7422, 97.92% with Desmonostoc_sp_SA25 and 97.99% with Desmonostoc_magnisporum_AR6_PS (Table 2). Results showed that Desmonostoc_alborizicum shared a ITS sequence similarity of 98.13% with Desmonostoc_salinum_CCM-UFV059, 97.97% with Desmonostoc_sp._111_CR4_BG11B, 98.70% with Desmonostoc_magnisporum_AR6_PS, 96.67% with Desmonostoc_sp._CCIBT_3489, 98.80% with Desmonostoc_sp._111_CR4_BG11N and 96.67% with Desmonostoc_sp._CCIBt3489_clone_53 (Table S4).
16S-23S rRNA ITS secondary structure.
Six reference sequences were used to search for ITS secondary structure. According to Johansen et al. , nine different areas (D1-D1’ helix, D2, D3, BOX B, BOX A, D4 and V3 helix) were found in the ITS secondary structure of studied strain. The D1-D1’ and Box-B regions of all studied strains were revealed to be very different in terms of length and shape (Fig. 5 and 6; Tables S6, S7 and S8). The D1-D1’ region included a terminal bilateral bulge (A), bilateral bulge (B), unilateral bulge (C), and basal clamp (D) (Fig. 5). The lengths of D1-D1’ helix varied from 65 nt (Desmonostoc magnisporum AR6_PS) to 69 nt (Desmonostocalborizicum, Desmonostoc sp. CCIBT 3489 and Desmonostoc sp. CCIBt3489 clone 53) (Table S5). The basal stem revealed to be the same for all studied strains (5’- GACCUA – UAGGUC- 3’), except for Desmonostoc sp. CCIBT 3489 and Desmonostoc sp. CCIBt3489 clone 53, which showed a different basal stem (5’ – GACCU- AGGUC 3’) (Fig. 5).
Box-B was nominated by Terminal Bilateral Bulge (A), Bilateral Bulge (B). Box-B helix was not found for Desmonostoc magnisporum AR6_PS and Desmonostoc sp. CCIBT 3489. As to the Box-B, lengths varied from 27 nt (Desmonostoc salinum CCM-UFV059, Desmonostoc sp. 111_CR4_BG11B and Desmonostoc sp. 111_CR4_BG11N) to 37 nt (Desmonostoc sp. CCIBt3489 clone 53), with studied strain showing a length of 31 nt (Fig. 6). Moreover V3 helix was only found for Desmonostoc sp. CCIBt3489 clone 53 and studied strain and was nominated like The D1-D1’ region. The V3 helix was similar in terms of length and shape between the studied strain and Desmonostoc sp. CCIBt3489 clone 53 (Fig. 7; Tables S7, S8 and S9).
Results of chemical analysis
Chemical analysis revealed the presence of at least four different microcystin (MC) variants (Table S9). The prominent variant in the sample was Microcystin-LR (MC-LR) in its standard as well in one of its demethylated forms ([D-Asp3] MC-LR). The other main variant was MC-RR (MC-RR) and one of its demethylated derivatives ([D-Asp3] MC-RR). The latter, however, was only present in trace amounts. The product ion spectra (MS2), retention times and molecular ion masses of MC-LR (Figure 8) and MC-RR were consistent with those of the measured standards. The MS2 spectra of the [D-Asp3] MC-RR and [D-Asp3] MC-LR were in agreement with fragmentation patterns described by Fujii et al. .