Microbial Endoliths on East Adriatic Limestone Coast: Morphological vs. Molecular Diversity

ABSTRACT

For the first time, a combined genotype-phenotype diversity analysis of endolithic microorganisms by pyrosequencing and microscopy was carried out across the intertidal and supratidal ranges of the Adriatic limestone coast. The coastal profiles represent ecotones between the sea and land, which express gradients of water supply, solar illumination, and salinity. The use of scanning electron microscopy imagery of resin casts showed that euendolith penetration remained shallow in the uppermost zones and was progressively deeper in the lower ranges where the water supply was more frequent. Microbial genotypes and phenotypes along the coastal profile showed large differences in dominance of taxa, but less in their diversity. The linkage between morphotypes observed along the profile and sequences was achieved by sequencing single cells/filaments, which were morphologically identified prior to their amplification. Molecular signatures of Hormathonema spp., Hyella caespitosa, Scytonema endolithicum, Solentia paulocellulare and Kyrtuthrix dalmatica were found on the profile and their specific placement was confirmed by morphological observations. Most sequences obtained were affiliated with Cyanobacteria (43%), Alphaproteobacteria (18%), and Gammaproteobacteria (7%). A total of 13 cyanobacterial morphospecies and 17 genotypes were found. Most cyanobacterial sequences belonged to the Pleurocapsales (33%), only 2% of the sequences belonged to the Nostocales and Oscillatoriales. The sequences corresponding to the cyanobacterial genus Hormathonema exhibited the highest abundances among all detected sequences, which is consistent with the microscopic observations in other parts of the world. The results of our investigation underline the importance of a combined approach in comparing the molecular data with microscopic observations in all phases of the study. Further RNA studies are needed to identify the actively growing parts of the endolithic community.

KEYWORDS:

• Adriatic Sea
• cyanobacteria
• endoliths
• pyrosequencing
• visualization of rock-boring microorganisms

Acknowledgments

We thank Dr Rosi Rippka for the discussions and suggestions, and Maria Brandes for help in DNA extraction. Special thanks are due to the Dive Center Bozava, Dugi Otok, Croatia.

Funding

Research was supported by the DFG project PA 842/9-1. International collaboration was supported by Alexander von Humboldt Foundation, Bonn, to S. Golubic and Hansewissenschaftskolleg (HWK), Institute for Advanced Studied in Delmenhorst, Germany for RA and his study group. This study was funded by the DFG (Deutsche Forschungsgemeinschaft, grant number PA 842/9-1).