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Cyanobacteria are adaptable and dominant organisms that exist in many harsh and extreme environments due to their great ecological tolerance. They produce various secondary metabolites, including cyanotoxins. While cyanobacteria are well studied in surface waters and some aerial habitats, numerous other habitats and niches remain underexplored. We collected 61 samples of: (i) biofilms from springs, (ii) aerial microbial mats from buildings and subaerial mats from caves, and (iii) water from borehole wells, caves, alkaline, saline, sulphidic, thermal, and iron springs, rivers, seas, and melted cave ice from five countries (Croatia, Georgia, Italy, Serbia, and Slovenia). We used (q)PCR to detect cyanobacteria (phycocyanin intergenic spacer-PC-IGS and cyanobacteria-specific 16S rRNA gene) and cyanotoxin genes (microcystins- mcyE , saxitoxins- sxtA , cylindrospermopsins- cyrJ ), as well as amplicon sequencing and morphological observations for taxonomic identification. Cyanobacteria were detected in samples from caves, a saline spring, and an alkaline spring. While mcyE or sxtA genes were not observed in any sample, cyrJ results showed the presence of a potential cylindrospermopsin producer in a biofilm from a sulphidic spring in Slovenia. This study contributes to our understanding of cyanobacteria occurrence in diverse habitats, including rare and extreme ones, and provides relevant methodological considerations for future research in such environments.

The effective identification of bacterial and fungal isolates is essential for microbiological monitoring in environments like speleotherapeutic caves. This study compares MALDI-TOF MS and the OmniLog ID System, two high-throughput culture-based identification methods. MALDI-TOF MS identified 80.0% of bacterial isolates to the species level, while the OmniLog ID System identified 92.9%. However, species-level matches between the methods were only 48.8%, revealing considerable discrepancies. For discrepant results, MALDI-TOF MS matched molecular identification at the genus level in 90.5% of cases, while the OmniLog ID System matched only in 28.6%, demonstrating MALDI-TOF MS's superiority. The OmniLog ID System had difficulties identifying genera from the order Micrococcales . Fungal identification success with MALDI-TOF MS was 30.6% at the species level, potentially improvable with a customised spectral library, compared to the OmniLog ID System's 16.7%. Metagenomic approaches detected around 100 times more microbial taxa than culture-based methods, highlighting human-associated microorganisms, especially Staphylococcus spp. In addition to Staphylococcus spp. and Micrococcus spp. as indicators of cave anthropisation, metagenomics revealed another indicator, Cutibacterium acnes . This study advocates a multi-method approach combining MALDI-TOF MS, the OmniLog ID System, culture-based, and metagenomic analyses for comprehensive microbial identification. Metagenomic sampling on nitrocellulose filters provided superior read quality and microbial representation over liquid sampling, making it preferable for cave air sample collection.

This study investigates the nitrate gradients within the deep biosphere of karst carbonate rocks and their resident microbiota. Samples were taken from borehole cores at depths down to 350 m below the surface, collected during geological site investigations for proposed railway tunnels and analysed using 16S rRNA amplicon sequencing. 16S rRNA amplicon sequencing analysis revealed relatively low microbial diversity, which can serve as a reliable indicator of the pristine nature of deep karst. However, some local hotspots of diversity are independent of depth. Pseudomonadota dominated the samples, with Gammaproteobacteria dominating at the class level. The low nitrate content in deep karst, in contrast to higher values closer to the surface, serves as an additional marker of its undisturbed and unpolluted status. Based on the prediction of functional profiles from 16S rRNA sequencing data, nitrates remain low due to indigenous microbial denitrification and assimilatory nitrate reduction. Pathways related to nitrogen fixation, ammonia assimilation, and nitrification were not confirmed. When elevated nitrate levels are observed in karst, they are most probably related to anthropogenic activities. Environmental factors other than depth and nitrate content play an important role in shaping bacterial communities.

Aerosols in caves are natural tracers and, together with climatic parameters, provide a detailed insight into atmospheric conditions, responses to climatic changes and anthropogenic influences in caves. Microbiological air monitoring in show caves is becoming increasingly useful to understand changes in cave ecosystems and to implement and review measures for sustainable cave use and tourism development. In 2017 and 2018, air along tourist trails in caves Postojnska jama and Škocjanske jame (Slovenia) was sampled before and after tourist visits. Samples were analysed using culture-dependent methods, flow cytometry, detection of β-D-glucan and lipopolysaccharide and compared with CO 2 and temperature data to measure anthropogenic influences and seasonality on aerobiota. While the presence of tourists significantly increased concentrations of airborne microorganisms ( p < 0.05), β-D-glucan and CO 2 did not show such a trend and were more dependent on seasonal changes. Locally, concentrations of cultivable microorganisms above 1000 CFU/m 3 were detected, which could have negative effects on the autochthonous microbiota and possibly on human health. A mixture of bacteria typically associated with humans was found in the air and identified with MALDI-TOF MS. Using MALDI-TOF MS, we achieved a 69.6% success rate in identification. Micrococcus luteus , Streptococcus mitis , Staphylococcus epidermidis and Moraxella spp. were recognized as good indicators of cave anthropisation.

Water suppliers should provide safe drinking water following preventive measures. This is especially important for karst water sources, as they are among the most vulnerable. Recently, there has been a strong focus on the early warning system, which mainly involves monitoring proxy parameters, but does not consider drainage area conditions and other monitoring recommendations. Here, we present an innovative strategy for assessing contamination risk of karst water sources that covers spatio-temporal dimensions and can be integrated into management practices. It is based on event-based monitoring and risk mapping and has been tested in a well-known study area. The holistic early warning system provides accurate spatial hazard and risk assessment and operational monitoring guidelines, including locations, indicator parameters, and temporal resolution and duration. In the study area, the high contamination risk, representing 0.5 % of the area, was spatially delineated. The highest probability of source contamination occurs during recharge events when proxy parameters such as bacteria, ATP, Cl, and Ca/Mg ratio should be monitored in addition to continuous monitoring of turbidity, EC, and T. Monitoring of sinking streams should serve as a preventive measure, since water transfer from ponors to springs has been shown to take about one day, and poor quality water is present for at least another day. Therefore, intensive monitoring should be conducted at intervals of a few hours for at least a week. Although hydrologic systems vary, the proposed strategy is particularly useful in systems where water flows rapidly and where remediation is not feasible., Competing Interests: Declaration of competing interest The authors of the manuscript certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

A natural cave environment subject to regular human visitation was selected for aerobiological study to minimize the effects of severe temperature fluctuations, UV radiation, and desiccation stress on the aerobiome. The longer sampling period of bioaerosols, up to 22 months, was generally not associated with a proportionally incremental and cumulative increase of microbial biomass. The culture-independent biomass indicator ATP enabled quick and reliable determination of the total microbial biomass. Total airborne microbial biomass was influenced by human visitation to the cave, as confirmed by significantly higher concentrations being observed along tourist footpaths ( p < 0.05). Airborne beta-glucans (BG) and lipopolysaccharide (LPS) are present in cave air, but their impact on the cave remains to be evaluated. Staphylococcus spp., as an indicator of human presence, was detected at all sites studied. Their long-term survival decrease is likely due to high relative humidity, low temperature, the material to which they adhere, and potentially natural elevated radon concentration. The most commonly recorded species were: S. saprophyticus , which was identified in 52% of the studied sites, S. equorum in 29%, and S. warneri in 24% of the studied sites. Only a few isolates were assigned to Risk group 2: S. aureus , S. epidermidis , S. haemolyticus , S. pasteuri , and S. saprophyticus .

Subterranean ecosystems are among the most widespread environments on Earth, yet we still have poor knowledge of their biodiversity. To raise awareness of subterranean ecosystems, the essential services they provide, and their unique conservation challenges, 2021 and 2022 were designated International Years of Caves and Karst. As these ecosystems have traditionally been overlooked in global conservation agendas and multilateral agreements, a quantitative assessment of solution-based approaches to safeguard subterranean biota and associated habitats is timely. This assessment allows researchers and practitioners to understand the progress made and research needs in subterranean ecology and management. We conducted a systematic review of peer-reviewed and grey literature focused on subterranean ecosystems globally (terrestrial, freshwater, and saltwater systems), to quantify the available evidence-base for the effectiveness of conservation interventions. We selected 708 publications from the years 1964 to 2021 that discussed, recommended, or implemented 1,954 conservation interventions in subterranean ecosystems. We noted a steep increase in the number of studies from the 2000s while, surprisingly, the proportion of studies quantifying the impact of conservation interventions has steadily and significantly decreased in recent years. The effectiveness of 31% of conservation interventions has been tested statistically. We further highlight that 64% of the reported research occurred in the Palearctic and Nearctic biogeographic regions. Assessments of the effectiveness of conservation interventions were heavily biased towards indirect measures (monitoring and risk assessment), a limited sample of organisms (mostly arthropods and bats), and more accessible systems (terrestrial caves). Our results indicate that most conservation science in the field of subterranean biology does not apply a rigorous quantitative approach, resulting in sparse evidence for the effectiveness of interventions. This raises the important question of how to make conservation efforts more feasible to implement, cost-effective, and long-lasting. Although there is no single remedy, we propose a suite of potential solutions to focus our efforts better towards increasing statistical testing and stress the importance of standardising study reporting to facilitate meta-analytical exercises. We also provide a database summarising the available literature, which will help to build quantitative knowledge about interventions likely to yield the greatest impacts depending upon the subterranean species and habitats of interest. We view this as a starting point to shift away from the widespread tendency of recommending conservation interventions based on anecdotal and expert-based information rather than scientific evidence, without quantitatively testing their effectiveness., (© 2022 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

A mixture of coal bottom ash and slag, with a fraction of fly ash (CAFAS) from steam locomotives, was placed in the cave Divaška jama to delimit and level tourist trails. Emplacement began in 1914 and carried on for several decades. The CAFAS mixed with other cave material gradually changed its structure and appearance. Currently the concentration of some elements in the CAFAS (As, Cu, Hg, Ni, Pb, Zn), and also to a lesser extent in cave sediments (Cr, Cu, Ni), indicates a possibly harmful effect on sediment-associated biota based on ecotoxicological assays. Compared to the cave sediment, the CAFAS contains distinctly different mineral phases and presents a different source of radioactivity. Microbial metabolic activity of CAFAS is low, 0.22 μl O 2 /gDW h, but higher than that of cave sediment. The present environmental hazards from CAFAS are estimated to be low. Whereas the emplacement of CAFAS was seen initially a long-term solution for waste disposal and management of the cave, it turned out that CAFAS enriches the underground environment with inorganic and organic compounds and disperses pollution into the cave ecosystem. After its removal from the cave, the CAFAS should be investigated thoroughly due to its susceptibility to alteration., (© 2021. The Author(s).)

Extremophiles inhabit a wide variety of environments. Here we focus on extremophiles in moderate climates in central Europe, and particularly in Slovenia. Although multiple types of stress often occur in the same habitat, extremophiles are generally combined into groups according to the main stressor to which they are adapted. Several types of extremophiles, e.g., oligotrophs, are well represented and diverse in subsurface environments and karst regions. Psychrophiles thrive in ice caves and depressions with eternal snow and ice, with several globally distributed snow algae and psychrophilic bacteria that have been discovered in alpine glaciers. However, this area requires further research. Halophiles thrive in salterns while thermophiles inhabit thermal springs, although there is little data on such microorganisms in central Europe, despite many taxa being found globally. This review also includes the potential use of extremophiles in biotechnology and bioremediation applications.

Relative humidity (RH) was measured at hourly intervals for approximately one year in two caves at seven stations near Playa del Carmen in Quintana Roo, Mexico. Sistema Muévelo Rico is a 1.1 km long cave with 12 entrances and almost no dark zone. Río Secreto (Tuch) is a large river cave with more than 40 km of passages, and an extensive dark zone. Given the need for cave specialists to adapt to saturated humidity, presumably by cuticular thinning, the major stress of RH would be its deviation from saturation. RH in Río Secreto (Tuch) was invariant at three sites and displayed short deviations from 100% RH at the other four sites. These deviations were concentrated at the end of the nortes and beginning of the rainy season. Three of the sites in Sistema Muévelo Rico showed a similar pattern although the timing of the deviations from 100% RH was somewhat displaced. Four sites in Sistema Muévelo Rico were more variable, and were analyzed using a measure of amount of time of deviation from 100% RH for each 24 hour period. Strong seasonality was evident but, remarkably, periods of constant high humidity were not the same at all sites. In most Sistema Muévelo Rico sites, there was a detectable 24 hour cycle in RH, although it was quite weak in about half of them. For Río Secreto (Tuch) only one site showed any sign of a 24 hour cycle. The troglomorphic fauna was more or less uniformly spread throughout the caves and did not concentrate in any one area or set of RH conditions. Compared to temperature, RH is much more constant, perhaps even more constant than the amount of light. However, changes in RH as a result of global warming may have a major negative effect on the subterranean fauna., Competing Interests: MCC Statistical Consulting LLC provided support in the form of salaries for CC. The commercial affiliation of MCC to MCC Statistical Consulting LLC does not alter our adherence to PLOS ONE policies on sharing data and materials.

Paradana is one of the biggest ice caves in Slovenia, with an estimated ice volume of 8,000 m 3 . Reflecting climatological conditions, the cave ice undergoes repeated freeze-thaw cycles and regular yearly deposition of fresh ice. Three distinct ice block samples, collected from the frozen lake in May 2016, were analysed to obtain data on ice physicochemical properties and the composition of associated microbiota. Isotopic composition of the ice samples ( 18 O, 2 H) and a local meteoric water line (LMWL) constructed for monthly precipitation at Postojna were used to estimate the isotopic composition of the water that formed the ice, which had high values of deuterium excess and low concentrations of chloride, sulphate and nitrate. The values of total organic carbon (1.93-3.95 mg/l) within the ice blocks fall within the range of those measured in karst streams. Total cell count in the ice was high and the proportion of cell viability increased along the depth gradient and ranged from 4.67 × 10 4 to 1.52 × 10 5 cells/ml and from 51.0 to 85.4%, respectively. Proteobacteria represented the core of the cave-ice microbiome (55.9-79.1%), and probably play an essential role in this ecosystem. Actinobacteria was the second most abundant phylum (12.0-31.4%), followed in abundance by Bacteroidetes (2.8-4.3%). Ice phylotypes recorded amounted to 442 genera, but only 43 genera had abundances greater than 0.5%. Most abundant were Pseudomonas, a well-known ice dweller, and Lysobacter, which previously was not reported in this context. Finally, two xanthophytes, Chloridella glacialis and Ellipsoidion perminimum, known from polar environments, were cultured from the ice. This indicates that the abundance and ecological role of phototrophs in such environments might be greater than previously deduced.

Local biodiversity trends over time are likely to be decoupled from global trends, as local processes may compensate or counteract global change. We analyze 161 long-term biological time series (15-91 years) collected across Europe, using a comprehensive dataset comprising ~6,200 marine, freshwater and terrestrial taxa. We test whether (i) local long-term biodiversity trends are consistent among biogeoregions, realms and taxonomic groups, and (ii) changes in biodiversity correlate with regional climate and local conditions. Our results reveal that local trends of abundance, richness and diversity differ among biogeoregions, realms and taxonomic groups, demonstrating that biodiversity changes at local scale are often complex and cannot be easily generalized. However, we find increases in richness and abundance with increasing temperature and naturalness as well as a clear spatial pattern in changes in community composition (i.e. temporal taxonomic turnover) in most biogeoregions of Northern and Eastern Europe.

Distributed environmental research infrastructures are important to support assessments of the effects of global change on landscapes, ecosystems and society. These infrastructures need to provide continuity to address long-term change, yet be flexible enough to respond to rapid societal and technological developments that modify research priorities. We used a horizon scanning exercise to identify and prioritize emerging research questions for the future development of ecosystem and socio-ecological research infrastructures in Europe. Twenty research questions covered topics related to (i) ecosystem structures and processes, (ii) the impacts of anthropogenic drivers on ecosystems, (iii) ecosystem services and socio-ecological systems and (iv), methods and research infrastructures. Several key priorities for the development of research infrastructures emerged. Addressing complex environmental issues requires the adoption of a whole-system approach, achieved through integration of biotic, abiotic and socio-economic measurements. Interoperability among different research infrastructures needs to be improved by developing standard measurements, harmonizing methods, and establishing capacities and tools for data integration, processing, storage and analysis. Future research infrastructures should support a range of methodological approaches including observation, experiments and modelling. They should also have flexibility to respond to new requirements, for example by adjusting the spatio-temporal design of measurements. When new methods are introduced, compatibility with important long-term data series must be ensured. Finally, indicators, tools, and transdisciplinary approaches to identify, quantify and value ecosystem services across spatial scales and domains need to be advanced., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The European cave spider, Meta menardi, is a representative of the troglophiles, i.e. non-strictly subterranean organisms. Our aim was to interpret the cytological results from an ecological perspective, and provide a synthesis of the hitherto knowledge about M. menardi into a theory of key features marking it a troglophile. We studied ultrastructural changes of the midgut epithelial cells in individuals spending winter under natural conditions in caves, using light microscopy and TEM. The midgut diverticula epithelium consisted of secretory cells, digestive cells and adipocytes. During winter, gradual vacuolization of some digestive cells appeared, and some necrotic digestive cells and necrotic adipocytes appeared. This cytological information completes previous studies on M. menardi starved under controlled conditions in the laboratory. In experimental starvation and natural winter conditions, M. menardi gradually exploit reserve compounds from spherites, protein granules and through autophagy, and energy-supplying lipids and glycogen, as do many overwintering arthropods. We found no special cellular response to living in the habitat. Features that make it partly adapted to the subterranean habitat include starvation hardiness as a possible preadaptation, an extremely opportunistic diet, a partly reduced orb, tracking and capturing prey on bare walls and partly reduced tolerance to below-zero temperatures.

Due to intrinsic characteristics of aquifers groundwater frequently passes between various types of aquifers without hindrance. The complex connection of underground water paths enables flow regardless of administrative boundaries. This can cause problems in water resources management. Numerical modelling is an important tool for the understanding, interpretation and management of aquifers. Useful and reliable methods of numerical modelling differ with regard to the type of aquifer, but their connections in a single hydrodynamic model are rare. The purpose of this study was to connect different models into an integrated system that enables determination of water travel time from the point of contamination to water sources. The worst-case scenario is considered. The system was applied in the Soča/Isonzo basin, a transboundary river in Slovenia and Italy, where there is a complex contact of karst and intergranular aquifers and surface flows over bedrock with low permeability. Time cell models were first elaborated separately for individual hydrogeological units. These were the result of numerical hydrological modelling (intergranular aquifer and surface flow) or complex GIS analysis taking into account the vulnerability map and tracer tests results (karst aquifer). The obtained cellular models present the basis of a contamination early-warning system, since it allows an estimation when contaminants can be expected to appear, and in which water sources. The system proves that the contaminants spread rapidly through karst aquifers and via surface flows, and more slowly through intergranular aquifers. For this reason, karst water sources are more at risk from one-off contamination incidents, while water sources in intergranular aquifers are more at risk in cases of long-term contamination. The system that has been developed is the basis for a single system of protection, action and quality monitoring in the areas of complex aquifer systems within or on the borders of administrative units., (Copyright © 2017 Elsevier B.V. All rights reserved.)