Characteristics of submerged and partially submerged caves (habitat type 8330) in Romania.

Underwater sea caves form a relatively under-examined habitat type within the marine regions of Europe, although they provide unique physical conditions such as reduced light and wave energy, in addition to reduced temperature amplitude. This study aimed at revealing the characteristics of submerged cavities on the southern Romanian continental shelf where six protected areas exist. We used high-resolution bathymetry data and side-scan sonar imaging to identify limestone outcrops where cavities would be most probable to form and then performed visual observation during SCUBA diving activities. We found that submerged cavities appear in all marine-protected areas and their neighboring unprotected areas from the shore to ~20-m depth mostly in the form of caverns, niches, and overhangs that meet the physical requirements of the habitat type 8330 as defined by the European Commission. We propose that habitat type 8330 should be listed in the Natura 2000 Standard Data Forms of the protected areas where it is missing, and we also propose that some protected areas should be expanded to include important areas with the occurrence of this habitat type. Finally, we note here for the first time the occurrence of Movile-type karst and an associated sulfidic water spring on the southern shore of Lake Techirghiol, similar to the region near Mangalia where it was first described in the literature. Although our finding prompts for a more detailed study, it indicates the existence of an underground ecosystem similar to that from the well-studied Movile Cave, but isolated by a distance of over 20 km.

Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave.

Microbial diversity in Movile Cave (Romania) was studied using bacterial and archaeal 16S rRNA gene sequence and functional gene analyses, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), soxB (sulfate thioesterase/thiohydrolase) and amoA (ammonia monooxygenase). Sulfur oxidizers from both Gammaproteobacteria and Betaproteobacteria were detected in 16S rRNA, soxB and RuBisCO gene libraries. DNA-based stable-isotope probing analyses using 13C-bicarbonate showed that Thiobacillus spp. were most active in assimilating CO2 and also implied that ammonia and nitrite oxidizers were active during incubations. Nitrosomonas spp. were detected in both 16S rRNA and amoA gene libraries from the 'heavy' DNA and sequences related to nitrite-oxidizing bacteria Nitrospira and Candidatus 'Nitrotoga' were also detected in the 'heavy' DNA, which suggests that ammonia/nitrite oxidation may be another major primary production process in this unique ecosystem. A significant number of sequences associated with known methylotrophs from the Betaproteobacteria were obtained, including Methylotenera, Methylophilus and Methylovorus, supporting the view that cycling of one-carbon compounds may be an important process within Movile Cave. Other sequences detected in the bacterial 16S rRNA clone library included Verrucomicrobia, Firmicutes, Bacteroidetes, alphaproteobacterial Rhodobacterales and gammaproteobacterial Xanthomonadales. Archaeal 16S rRNA sequences retrieved were restricted within two groups, namely the Deep-sea Hydrothermal Vent Euryarchaeota group and the Miscellaneous Crenarchaeotic group. No sequences related to known sulfur-oxidizing archaea, ammonia-oxidizing archaea, methanogens or anaerobic methane-oxidizing archaea were detected in this clone library. The results provided molecular biological evidence to support the hypothesis that Movile Cave is driven by chemolithoautotrophy, mainly through sulfur oxidation by sulfur-oxidizing bacteria and reveal that ammonia- and nitrite-oxidizing bacteria may also be major primary producers in Movile Cave.