Genotypic and morphological diversity of cyanobacteria in the Rupite hot spring (Bulgaria) was investigated by means of optical microscopy, cultivation, single-cell PCR, and 16S rRNA gene amplicon sequencing. Altogether, 34 sites were investigated along the 71-39 °C temperature gradient. Analysis of samples from eight representative sites shown that Illumina, optical microscopy, and Roche 454 identified 72, 45 and 19% respective occurrences of all cumulatively present taxa. Optical microscopy failed to detect species of minor occurrence; whereas, amplicon sequencing technologies suffered from failed primer annealing and the presence of species with extensive extracellular polysaccharides production. Amplicon sequencing of the 16S rRNA gene V5-V6 region performed by Illumina identified the cyanobacteria most reliably to the generic level. Nevertheless, only the combined use of optical microscopy, cultivation and sequencing methods allowed for reliable estimate of the cyanobacterial diversity. Here, we show that Rupite hot-spring system hosts one of the richest cyanobacterial flora reported from a single site above 50 °C. Chlorogloeopsis sp. was the most abundant at the highest temperature (68 °C), followed by Leptolyngbya boryana, Thermoleptolyngbya albertanoae, Synechococcus bigranulatus, Oculatella sp., and Desertifilum sp. thriving above 60 °C, while Leptolyngbya geysericola, Geitlerinema splendidum, and Cyanobacterium aponinum were found above 50 °C.

Center Algatech Institute of Microbiology CAS 37981 Třeboň Czech Republic

Department of Plant and Environmental Science Norwegian University of Life Sciences 1432 Ås Norway

Department of Plant Ecology Institute of Botany CAS 37901 Třeboň Czech Republic

Faculty of Science University of South Bohemia 37005 České Budějovice Czech Republic

Group Microbial Communication Helmholtz Centre for Infection Research 38124 Brunswick Germany

Group Microbial Interactions and Processes Helmholtz Centre for Infection Research 38124 Brunswick Germany

Institute of Aquaculture CENAKVA Faculty of Fisheries and Protection of Waters University of South Bohemia 37005 České Budějovice Czech Republic

Institute of Plant Physiology and Genetics BAS 1113 Sofia Bulgaria

Israel Oceanographic and Limnological Research Ltd National Center for Mariculture 88112 Eilat Israel

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