Sulfuric Caves Research Articles

Exploring Biodiversity and Food Webs in Sulfur Cave in the Vromoner Canyon on the Greek–Albanian Border

Sulfidic caves support diverse and abundant subterranean communities, including numerous endemic species and complex food webs, though the full extent of species diversity and resource utilization in these ecosystems remains largely unexplored. This paper presents the results of biological surveys conducted from 2023 to 2024 in Sulfur Cave, located in the Vromoner Canyon on the Greek–Albanian border, focusing on microbial, vertebrate, and invertebrate communities and investigating the structure of the subterranean food web. The microbial communities from the different biofilms are dominated by chemosynthetic sulfur-oxidizing microorganisms, specifically filamentous bacteria such as Thiotrix and Beggiatoa. Two species of fish, an eel (Anguilla sp.) and a Cyprinid (Alburnoides sp.), and six bat species from three families (Rhinolophidae, Miniopteridae, and Vespertilionidae) were documented. The invertebrate fauna includes five aquatic species, 25 terrestrial species, and four amphibiotic species. Among these, eight species are endemic, and seven species exhibit troglomorphic traits. Stable isotope analysis showed light carbon and nitrogen values for the terrestrial and aquatic invertebrates, suggesting that subterranean communities rely on food produced in situ by chemoautotrophic microorganisms. Our results identified cave areas of significant biological relevance and provided reference data to inform conservation actions aimed at preserving the biodiversity of this sulfidic cave.

The Shoshone Canyon Conduit Cave: A Greater Yellowstone Ecosystem Sulfur Cave

Fve miles west of Cody, Wyoming and ~25 miles east of Yellowstone National Park lies the Shoshone Canyon Conduit Cave (Fig. 1, Suppl. material 1). Bisecting Cedar Mountain, an irrigation tunnel built by the Bureau of Reclamation (BoR), managed by the Heart Mountain Irrigation District, delivers water from the BoR Buffalo Bill Reservoir to more than 30,000 acres of irrigated lands in and around Cody. During the construction of that tunnel in the late 1940’s, a cave was discovered and the tunnel was made to travel though the cave with only light disturbance to the cave. The cave can only be accessed with permission in the non-irrigation time of year. The cave is rich in sulfides and sulfur deposits with unique speleothems. To date, we have conducted a preliminary geobiological survey of the geochemistry, microbiology and mineralogy of this sulfur cave on its speleothems, mineral deposits and waters. Sampled waters, of which there is little, were all highly acidic (pH < 2). Microbiologically, an analysis of microbial communities present in approximately 25 sample locations (Fig. 1) to answer the question of—who is there?—was conducted via small subunit ribosomal 16S rRNA gene (for Bacteria and Archaea) and 18S rRNA (for Eukarya) analyses, prepared using a polymerase chain reaction (PCR) primer-pair that allows for the even amplification of all three domains of life. All samples were relatively low in biomass and resultant community analysis indicates a variety of Bacteria and Archaea phyla are represented with a dominance of known sulfur metabolizers. Mineralogically, petrography reveals a variety of crystal growth and habit in this sulfur-dominated, calcium carbonate-driven karstic ecosystem. X-ray diffraction analysis (XRD) was used to better determine the kinds and extant of mineral morphotypes and were surprisingly variable. The Shoshone Canyon Conduit Cave is a most intriguing sulfur cave to reveal what is known of the geobiology of sulfur caves in the Rocky Mountain Region. Findings from this work will likely apply to other cave systems such as Villa Luz (Mexico) and the Frasassi system (Italy). Finally, to learn about the Cedar Mountain Caves will inform on how either or both the National Park Service (NPS) and Bureau of Land Management (BLM) may better manage them as a meaningful component of the Greater Yellowstone Ecosystem.

The fascinating biology of stinky caves

The discovery of Movile Cave in 1986, similarly to the discovery of the deep-sea vents 9 years earlier, showed that, when redox interfaces are present in subsurface ecosystems, diverse and abundant biological communities can thrive based primarily on autochthonous food produced in situ by chemosynthesis. Additional caves, partially flooded by sulfidic water have been discovered, i.e. the Frasassi caves in Italy, Ein-Nur Cave and Ayyalon Cave in Israel, Melissotrypa Cave in Greece, Tashan Cave in Iran, caves in the Sharo-Argun Valley in the Caucasus Mountains, etc. All of these are inhabited by numerous endemic cave-adapted organisms present in dense populations sustained by a rich food base generated within the subterranean environment by microorganisms that use atmospheric oxygen to oxidize the reduced chemical compounds present in the water (H2S, NH4+, CH4). Stable isotope data has shown that the biological communities found in sulfidic caves are not dependent on carbon fixation in green plants that use light energy in the process of photosynthesis. Special adaptations have been identified in some of these inhabitants that allow them to cope with the adverse environmental conditions such as toxicity of H2S, hypoxia, and extreme pH values. Symbiotic associations between crustaceans, protozoa, and sulfide oxidizing bacteria were discovered in several sulfidic caves. New species of microorganisms have also been described from these unusual subsurface environments. Unusual microbiomes have recently been discovered in dry caves that contain chemoclines between heavy volcanic gasses dominated by CO2, but also containing H2S and CH4, and atmospheric air that floats on top of the volcanic gas emissions. Unique sulfur mineralization, microbial biofilms containing new species of bacteria that thrive at extremely pH values, symbiotic relationships between bacteria and fungi have been discovered in Sulfur Cave in Romania, Bosoletto Cave in Italy, and Sousaki caves in Greece. The extreme life conditions encountered in these caves are of special interest to astrobiologists using these caves as a model for better understanding conditions that life may be present in on other planets and celestial bodies in our Solar System and beyond.

Sulfated glycosaminoglycan-like polymers are present in an acidophilic biofilm from a sulfidic cave

Sulfated glycosaminoglycans (sGAG) are negatively charged extracellular polymeric substances that occur in biofilms from various environments. Yet, it remains unclear whether these polymers are acquired from the external environment or produced by microbes in the biofilm. To resolve this, we analyzed the presence of sGAGs in samples of an acidophilic biofilm collected from Sulfur Cave in Puturosu Mountain (Romania), an environment that is largely inaccessible to contamination. A maximum of 55.16 ± 2.06 μg sGAG-like polymers were recovered per mg of EPS. Enzymatic treatment with chondroitinase ABC resulted in a decrease of the mass of these polymers, suggesting the structure of the recovered sGAG is similar to chondroitin. Subsequent FT-IR analysis of these polymers revealed absorbance bands at 1230 cm−1, 1167 cm−1 and 900 cm−1, indicating a possible presence of polysaccharides and sulfate. Analysis of genomic sequences closely related to those predominant in the acidophilic biofilm, contained genes coding for sulfotransferase (an enzyme needed for the production of sGAG), which supports the hypothesis of microbial synthesis of sGAGs within the biofilm.

Sulfur Cave (Romania), an extreme environment with microbial mats in a CO2-H2S/O2 gas chemocline dominated by mycobacteria

Sulfur Cave (Puturosu Mountain, Romania) is an extreme environment, unique for displaying life in a gas chemocline. The lower part of the cave is filled with CO2, CH4, and H2S of mofettic origin, while the upper part contains air that floats above the heavier volcanic gasses. S° and H2SO4 (from sulfur-oxidation) cover the cave wall at and below the CO2-H2S:O2 gas/gas interface. On the cave wall, near the interface the pH is <1 and unusual microbial biofilms occur on the rock’s surface. We provide context information on the geology, mineralogy, chemistry and biology to better understand this unique environment. We have used X-ray diffraction, optical microscopy, scanning electron microscopy with EDAX capabilities, stable isotope analysis and 16S and 18S rDNA amplicon sequencing. The most common taxa in the microbial biofilms are Mycobacteria, Acidithiobacillus and Ferroplasmaceae. Liquid water in this system originates solely from condensation of water vapor onto the cave walls making inflow of organic carbon from outside unlikely. The most likely primary source of energy for this microbial community is sulfur oxidation with H2S and S° as main reductants and atmospheric O2 as the main oxidant. Ferric iron from the rock surface is another potential oxidant. In Sulfur Cave, gaseous CO2 (from mofettic emission) maintains the stability of the gas chemocline. Sulfur Cave biofilms can help the search for extreme life in the subsurface, near volcanic systems on Earth and Mars. The Sulfur Cave example shows that a habitable environment can be established underground in gas chemoclines near CO2-dominated gas discharge zones, where it can have a steady supply of water and energy.

Toxic hydrogen sulfide and dark caves: life‐history adaptations in a livebearing fish (Poecilia mexicana, Poeciliidae)

Life-history traits are very sensitive to extreme environmental conditions, because resources that need to be invested in somatic maintenance cannot be invested in reproduction. Here we examined female life-history traits in the Mexican livebearing fish Poecilia mexicana from a variety of benign surface habitats, a creek with naturally occurring toxic hydrogen sulfide (H2S), a sulfidic cave, and a non-sulfidic cave. Previous studies revealed pronounced genetic and morphological divergence over very small geographic scales in this system despite the absence of physical barriers, suggesting that local adaptation to different combinations of two selection factors, toxicity (H2S) and darkness, is accompanied by very low rates of gene flow. Hence, we investigated life-history divergence between these populations in response to the selective pressures of darkness and/or toxicity. Our main results show that toxicity and darkness both select for (or impose constraints on) the same female trait dynamics: reduced fecundity and increased offspring size. Since reduced fecundity in the sulfur cave population was previously shown to be heritable, we discuss how divergent life-history evolution may promote further ecological divergence: for example, reduced fecundity and increased offspring autonomy are clearly beneficial in extreme environments, but fish with these traits are outcompeted in benign habitats.

Male-biased predation of a cave fish by a giant water bug

Male-biased predation has been described from several epigean species, and in many cases, intrinsic differences between the sexes (such as male ornaments) have been suggested as an explanation. Here we report on male-biased predation of a cave fish (Poecilia mexicana) by an aquatic insect (Belostoma sp.) in a Mexican sulfur cave. P. mexicana use aquatic surface respiration (ASR) to survive in their sulfidic, hypoxic habitat. We found that males typically exhibit more ASR activity than females, which leads to increased exposure to the sit-and-wait predator that catches fish near the water surface. Our finding is novel, because male vulnerability to predation is not directly related to male traits involved in courtship, but rather due to other sexual differences in behavior and ultimately, oxygen demands.

A new and morphologically distinct population of cavernicolous Poecilia mexicana (Poeciliidae: Teleostei)

The cave molly, Poecilia mexicana, from the Cueva del Azufre, a sulfur cave in Tabasco, Mexico, ranks among the best-studied cave fishes worldwide, despite being known from a single population only. Here we describe a newly discovered second population of cave-dwelling P. mexicana from a nearby, but mostly non-sulfidic cave (Luna Azufre). Despite apparent similarities between the two populations (such as reduced eye diameter and reduced pigmentation), a geometric morphometric analysis revealed pronounced morphological differentiation between the two cave forms.

Subsurface sulfur systems: Production and preservation of distinctive biogenic signatures in sulfur, iron, manganese, and carbonate cave systems

Subsurface sulfur systems: Production and preservation of distinctive biogenic signatures in sulfur, iron, manganese, and carbonate cave systems

Choosy males from the underground: male mating preferences in surface- and cave-dwelling Atlantic mollies (Poecilia mexicana)

Atlantic mollies (Poecilia mexicana) inhabit a variety of surface habitats, but they also occur in a sulfur cave in southern Mexico. We examined male mate choice relative to female body size in the cave population and in the most closely related surface-dwelling population from a nearby river. Males from both populations were either light- or dark-reared and could choose between two differently sized females either on the basis of visual cues in light or on the basis of solely nonvisual cues in darkness. Sexual preferences were estimated from the degree of association. Cave molly males always showed a preference for the larger female, both in light and in darkness. Among the surface males, only light-reared males showed a preference in the visual cues test, but not in darkness. In a control experiment, we demonstrated that male association preferences directly translate into actual mating preferences. Apparently, using visual cues for mate choice is the ancestral state in this system, and using nonvisual cues has evolved as a novel trait in the cave population. We discuss the evolution of nonvisual male mate choice in the context of changed environmental conditions, namely the absence of light, hypoxia, and toxic hydrogen sulfide in the cave.