Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern

Abstract

Bacterial and archaeal assemblages have been studied in a multipond solar saltern using a range of microbial ecology techniques by four laboratories simultaneously. These include 16S rDNA sequencing from both denaturing gradient gel electrophoresis (DGGE) and clone libraries, and culturing methods. Water samples from eight ponds were analysed, covering a salinity range from near sea water (4% salt) to saturated sodium chloride (37% salt; ponds called crystallizers). Clone libraries focused on ponds with salinity of 8%, 22% and 32%. Although different cloning strategies were able to retrieve the same type of dominant sequences, there were differing degrees of success with less abundant sequences. Thus, the use of two sets of primers recovered a higher number of phylotypes. Bacterial and archaeal isolates were, however, different from any of the retrieved environmental sequences. For Bacteria, most sequences in the 8% salt pond were related to organisms of marine origin. Thus, representatives of the alpha-, beta-, gamma- and epsilon-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides group (CFB), high-G+C Gram-positive bacteria and cyanobacteria were found. In the 22% salt pond, alpha- and gamma-Proteobacteria, cyanobacteria and CFB were the only groups found, and most of them were related to specialized halophilic bacteria. From the 32% salt pond, only CFB were found, and most of the sequences retrieved clustered with Salinibacter ruber, an extremely halophilic bacterium. A decrease in the richness of bacterial genera was therefore apparent along the gradient. Archaea behaved quite similarly. In the lowest salinity ponds, sequences were related to environmental clones of Marine Archaea Group III (Thermoplasmales relatives) and to unclassified branches of Euryarchaeaota. In the 8%, 22% and 32% ponds, most of the clones were related to different cultured strains of Halobacteriaceae. Finally, most sequences from the crystallizers clustered with the uncultured square archaeon SPhT. Crenarchaeaota were not detected. Despite the fact that higher prokaryotic richness was apparent in the lower salinity ponds than in the crystallizers, the diversity index from clone libraries calculated according to Shannon and Weaver did not show this trend. This was because diversity in the crystallizers can be considered as 'microdiversity', the co-existence of several closely related clones of Bacteria (the S. ruber cluster) and Archaea (the SPhT cluster). Regardless of the changes in abundance, both Bacteria and Archaea showed the same pattern; as salinity increased, the number of different clusters decreased, and only one cluster became dominant. Both clusters, however, showed a considerable degree of microdiversity. The meaning of such microdiversity remains to be determined.