Groundwater is a hidden global keystone ecosystem.

Groundwater is a vital ecosystem of the global water cycle, hosting unique biodiversity and providing essential services to societies. Despite being the largest unfrozen freshwater resource, in a period of depletion by extraction and pollution, groundwater environments have been repeatedly overlooked in global biodiversity conservation agendas. Disregarding the importance of groundwater as an ecosystem ignores its critical role in preserving surface biomes. To foster timely global conservation of groundwater, we propose elevating the concept of keystone species into the realm of ecosystems, claiming groundwater as a keystone ecosystem that influences the integrity of many dependent ecosystems. Our global analysis shows that over half of land surface areas (52.6%) has a medium-to-high interaction with groundwater, reaching up to 74.9% when deserts and high mountains are excluded. We postulate that the intrinsic transboundary features of groundwater are critical for shifting perspectives towards more holistic approaches in aquatic ecology and beyond. Furthermore, we propose eight key themes to develop a science-policy integrated groundwater conservation agenda. Given ecosystems above and below the ground intersect at many levels, considering groundwater as an essential component of planetary health is pivotal to reduce biodiversity loss and buffer against climate change.

Microbial Ecosystems in Movile Cave: An Environment of Extreme Life.

Movile Cave, situated in Romania close to the Black Sea, constitutes a distinct and challenging environment for life. Its partially submerged ecosystem depends on chemolithotrophic processes for its energetics, which are fed by a continuous hypogenic inflow of mesothermal waters rich in reduced chemicals such as hydrogen sulfide and methane. We sampled a variety of cave sublocations over the course of three years. Furthermore, in a microcosm experiment, minerals were incubated in the cave waters for one year. Both endemic cave samples and extracts from the minerals were subjected to 16S rRNA amplicon sequencing. The sequence data show specific community profiles in the different subenvironments, indicating that specialized prokaryotic communities inhabit the different zones in the cave. Already after one year, the different incubated minerals had been colonized by specific microbial communities, indicating that microbes in Movile Cave can adapt in a relatively short timescale to environmental opportunities in terms of energy and nutrients. Life can thrive, diversify and adapt in remote and isolated subterranean environments such as Movile Cave.

Karst spring microbiome: Diversity, core taxa, and community response to pathogens and antibiotic resistance gene contamination.

Karst aquifers are important water resources for drinking water supplies worldwide. Although they are susceptible to anthropogenic contamination due to their high permeability, there is a lack of detailed knowledge on the stable core microbiome and how contamination may affect these communities. In this study, eight karst springs (distributed across three different regions in Romania) were sampled seasonally for one year. The core microbiota was analysed by 16S rRNA gene amplicon sequencing. To identify bacteria carrying antibiotic resistance genes and mobile genetic elements, an innovative method was applied, consisting of high-throughput antibiotic resistance gene quantification performed on potential pathogen colonies cultivated on Compact Dry™ plates. A taxonomically stable bacterial community consisting of Pseudomonadota, Bacteroidota, and Actinomycetota was revealed. Core analysis reaffirmed these results and revealed primarily freshwater-dwelling, psychrophilic/psychrotolerant species affiliated to Rhodoferax, Flavobacterium, and Pseudomonas genera. Both sequencing and cultivation methods indicated that more than half of the springs were contaminated with faecal bacteria and pathogens. These samples contained high levels of sulfonamide, macrolide, lincosamide and streptogramins B, and trimethoprim resistance genes spread primarily by transposase and insertion sequences. Differential abundance analysis found Synergistota, Mycoplasmatota, and Chlamydiota as suitable candidates for pollution monitoring in karst springs. This is the first study highlighting the applicability of a combined approach based on high-throughput SmartChip™ antibiotic resistance gene quantification and Compact Dry™ pathogen cultivation for estimating microbial contaminants in karst springs and other challenging low biomass environments.

Cave Thiovulum (Candidatus Thiovulum stygium) differs metabolically and genomically from marine species.

Thiovulum spp. (Campylobacterota) are large sulfur bacteria that form veil-like structures in aquatic environments. The sulfidic Movile Cave (Romania), sealed from the atmosphere for ~5 million years, has several aqueous chambers, some with low atmospheric O2 (~7%). The cave's surface-water microbial community is dominated by bacteria we identified as Thiovulum. We show that this strain, and others from subsurface environments, are phylogenetically distinct from marine Thiovulum. We assembled a closed genome of the Movile strain and confirmed its metabolism using RNAseq. We compared the genome of this strain and one we assembled from public data from the sulfidic Frasassi caves to four marine genomes, including Candidatus Thiovulum karukerense and Ca. T. imperiosus, whose genomes we sequenced. Despite great spatial and temporal separation, the genomes of the Movile and Frasassi Thiovulum were highly similar, differing greatly from the very diverse marine strains. We concluded that cave Thiovulum represent a new species, named here Candidatus Thiovulum stygium. Based on their genomes, cave Thiovulum can switch between aerobic and anaerobic sulfide oxidation using O2 and NO3- as electron acceptors, the latter likely via dissimilatory nitrate reduction to ammonia. Thus, Thiovulum is likely important to both S and N cycles in sulfidic caves. Electron microscopy analysis suggests that at least some of the short peritrichous structures typical of Thiovulum are type IV pili, for which genes were found in all strains. These pili may play a role in veil formation, by connecting adjacent cells, and in the motility of these exceptionally fast swimmers.

Database of Romanian cave invertebrates with a Red List of cave species and a list of hotspot/coldspot caves.

BACKGROUND: The increasing human impact in Romanian caves raises the urgency of publishing a correct database of the strictly-adapted cave fauna. Previous attempts at indexing cave fauna and classifying caves by using their fauna opened many questions regarding the use of an incomplete list of cave species and mixed lists of troglobionts/stygobionts with troglophiles/stygophiles for ranking caves with priority for protection. It has also become obvious that there is a need to publish a list of Romanian cave species that are under threat. Cave species in Romania (and elsewhere) are endemic on small ranges, are unique and must be considered as important units for conservation. A cave must be equally protected if it has one or more rare and strictly endemic cave species. Although not exhaustive, we here provide the first checklist of Romanian troglobionts/stygobionts developed in the framework of the DARKFOOD and GROUNDWATERISK projects, coordinated by the "Emil Racovita" Institute of Speleology, Cluj-Napoca, Romania. The GIS application was used to complement the checklist of cave species with data on caves and surface environments above the caves. Until complete data on species diversity and population sizes are made available for each cave, measures of conservation can be implemented, based on the presence/absence of cave species, while classifications of caves for protection, based on the number of species, must be avoided. We also propose a list of Romanian caves with fauna that are under threat and a tentative Red List of Romanian troglobiont/stygobionts. NEW INFORMATION: This is the first database with identified troglobiont and stygobiont species of Romania, with a critical analysis of their distribution inside the country. A list of caves that need protection for their rare and unique species and a tentative Red List of Romanian cave fauna are also added. A total of 173 species were identified, of which 77 troglobionts and 96 stygobionts are currently registered in 366 caves. The database is divided into two parts, one part with a list of troglobionts, their revised systematic position, cave name, cave code and geographic region; and the second part with the same information on stygobionts. The database represents the contribution of many active researchers, who are the authors of this paper and of review publications of many other authors of the "Emil Racovița" Institute of Speleology.

Fungi in perennial ice from Scarișoara Ice Cave (Romania).

Screening of 1,000-years old ice layers from the perennial ice block of Scarișoara Ice Cave (NW Romania) revealed the presence of fungal communities. Using culture-dependent methods and molecular techniques based on DGGE fingerprinting of 18S rRNA gene fragments and sequencing, we identified 50 cultured and 14 uncultured fungi in presently-forming, 400 and 900 years old ice layers, corresponding to 28 distinct operational taxonomic units (OTUs). The dominant ice-contained fungal OTUs were related to Ascomycota, Basidiomycota and Cryptomycota phyla. Representatives of Mucoromycota and Chytridiomycota were also isolated from recent and 400 years old ice samples. The cryophilic Mrakia stokesii was the most abundant fungal species found in the cave ice samples of all prospected ages, alongside other cryophilic fungi also identified in various glacial environments. Ice deposits formed during the Little Ice Age (dated between AD 1,250 and 1,850) appeared to have a higher fungal diversity than the ice layer formed during the Medieval Warm Period (prior to AD 1,250). A more complex fungal community adapted to low temperatures was obtained from all analyzed ice layers when cultivated at 4 °C as compared to 15 °C, suggesting the dominance of cold-adapted fungi in this glacial habitat. The fungal distribution in the analyzed cave ice layers revealed the presence of unique OTUs in different aged-formed ice deposits, as a first hint for putative further identification of fungal biomarkers for climate variations in this icy habitat. This is the first report on fungi from a rock-hosted cave ice block.

Niphargus-Thiothrix associations may be widespread in sulphidic groundwater ecosystems: evidence from southeastern Romania.

Niphargus is a speciose amphipod genus found in groundwater habitats across Europe. Three Niphargus species living in the sulphidic Frasassi caves in Italy harbour sulphur-oxidizing Thiothrix bacterial ectosymbionts. These three species are distantly related, implying that the ability to form ectosymbioses with Thiothrix may be common among Niphargus. Therefore, Niphargus-Thiothrix associations may also be found in sulphidic aquifers other than Frasassi. In this study, we examined this possibility by analysing niphargids of the genera Niphargus and Pontoniphargus collected from the partly sulphidic aquifers of the Southern Dobrogea region of Romania, which are accessible through springs, wells and Movile Cave. Molecular and morphological analyses revealed seven niphargid species in this region. Five of these species occurred occasionally or exclusively in sulphidic locations, whereas the remaining two were restricted to nonsulphidic areas. Thiothrix were detected by PCR on all seven Dobrogean niphargid species and observed using microscopy to be predominantly attached to their hosts' appendages. 16S rRNA gene sequences of the Thiothrix epibionts fell into two main clades, one of which (herein named T4) occurred solely on niphargids collected in sulphidic locations. The other Thiothrix clade was present on niphargids from both sulphidic and nonsulphidic areas and indistinguishable from the T3 ectosymbiont clade previously identified on Frasassi-dwelling Niphargus. Although niphargids from Frasassi and Southern Dobrogea are not closely related, the patterns of their association with Thiothrix are remarkably alike. The finding of similar Niphargus-Thiothrix associations in aquifers located 1200 km apart suggests that they may be widespread in European groundwater ecosystems.

Spatial heterogeneity in sediment-associated bacterial and eukaryotic communities in a landfill leachate-contaminated aquifer.

Heterogeneity in eukaryotic and bacteria community structure in surface and subsurface sediment samples downgradient of the Banisveld landfill (The Netherlands) was studied using a culturing-independent molecular approach. Along a transect covering the part of the aquifer most polluted by landfill leachate, sediment was sampled at 1-m depth intervals, until a depth of 5.5 m, at four distances from the landfill. Two drillings were placed in a nearby clean area as a reference. Denaturing gradient gel electrophoresis banding patterns revealed high bacterial and eukaryotic diversity and complex community structures. Bacteria and eukaryotic community profiles in polluted samples grouped different from those in clean samples. Bacteria community profiles in surface samples clustered together and separately from subsurface community profiles. Subsurface bacteria profiles clustered in a location-specific manner. Eukaryotic community structure did not significantly relate to distance from the landfill or depth. No significant spatial autocorrelation of bacteria or eukaryotic communities was observed over 1-m depth intervals per sampling location. Spatial heterogeneity in sediment-associated bacterial communities appears to be much larger than in groundwater. We discuss how on the one hand, spatial heterogeneity may complicate the assessment of microbial community structure and functioning, while on the other it may provide better opportunities for natural attenuation.

Eukaryotic diversity in an anaerobic aquifer polluted with landfill leachate.

Eukaryotes may influence pollutant degradation processes in groundwater ecosystems by activities such as predation on bacteria and recycling of nutrients. Culture-independent community profiling and phylogenetic analysis of 18S rRNA gene fragments, as well as culturing, were employed to obtain insight into the sediment-associated eukaryotic community composition in an anaerobic sandy aquifer polluted with landfill leachate (Banisveld, The Netherlands). The microeukaryotic community at a depth of 1 to 5 m below the surface along a transect downgradient (21 to 68 m) from the landfill and at a clean reference location was diverse. Fungal sequences dominated most clone libraries. The fungal diversity was high, and most sequences were sequences of yeasts of the Basidiomycota. Sequences of green algae (Chlorophyta) were detected in parts of the aquifer close (<30 m) to the landfill. The bacterium-predating nanoflagellate Heteromita globosa (Cercozoa) was retrieved in enrichments, and its sequences dominated the clone library derived from the polluted aquifer at a depth of 5 m at a location 21 m downgradient from the landfill. The number of culturable eukaryotes ranged from 10(2) to 10(3) cells/g sediment. Culture-independent quantification revealed slightly higher numbers. Groundwater mesofauna was not detected. We concluded that the food chain in this polluted aquifer is short and consists of prokaryotes and fungi as decomposers of organic matter and protists as primary consumers of the prokaryotes.