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Fig. S1. Archaeal 16S rRNA gene phylogenetic tree based on phylogenetic analysis of all clones recovered from three sublayers of the Thetis Lake. The tree was constructed by neighbour-joining method and Jukes–Cantor distance matrix using the ARB software. Bootstrap values > 50 and ≥ 70 are evidenced by open and filled circles at the nodes, respectively, and were calculated over 1000 random repetitions. Sequences obtained in this study are indicated in bold.

Fig. S2. Bacterial 16S rRNA gene phylogenetic tree based on phylogenetic affiliation of all clones recovered from three sublayers of the Thetis Lake. The tree was constructed by neighbour-joining method and Jukes–Cantor distance matrix using the ARB software. Bootstrap values > 50 and ≥ 70 are shown as empty and filled circles, respectively, and were calculated over 1000 random repetitions. Sequences obtained in this study are indicated in bold.

Fig. S3. Comparison between biodiversity data obtained from the three different depths, analysed by DGGE (UPGMA) and clone library (LIBSHUFF method). DGGE data of bacterial (left) and archaeal (right) communities were elaborated in UPGMA cluster analysis. Bray Curtis similarity was applied on a matrix constructed taking into account the presence or absence of the individual bands obtained from the DGGE profiles. Values indicated under the UPGMA tree are the statistical difference (delta-C) in the compositions of the clone libraries obtained by LIBSHUFF method. Each comparison was statistically significant (P-value of 0.001).

Fig. S4. Pairwise comparison of the Thetis Lake phylotypes (cut-off > 97% similarity) with those of other thalassohaline Mediterranean DHALs studied so far. Left column means that three bacterial and nine archaeal phylotypes were found only in Thetis Lake (Thetis-specific), whereas other columns demonstrated the number of phylotypes shared between Thetis-L’Atalante, Thetis-Bannock and Thetis-Urania brine lakes respectively.

Fig. S5. Phylogenetic affiliation of the monophyletic CbbLA aa (A) and aclA (B) nucleotide sequences as determined by neighbour-joining analysis/Poisson correction of distances and by neighbour-joining method/Jukes–Cantor distance matrix using the MEGA software respectively. The scale bar represents 2% amino acid (A) and 0.05 nucleotide changes per nucleotide position (B). Bootstrap values > 50 and ≥ 70 are shown as open and filled circles, respectively, and were calculated over 1000 random repetitions. Sequences obtained in this study are indicated in bold.

Table S1. Primer sequences used in this study.

Table S2. Diversity indices calculated for the six clone libraries generated by DOTUR software. T1-3 corresponds to the layers sampled in Thetis Lake; A and B stand for Archaea and Bacteria clone libraries respectively.

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