Background: Movile Cave (Dobrogea, SE Romania) hosts a subterranean chemoautotrophically-based ecosystem supported by a sulfidic thermal aquifer analogous to the deep-sea hydrothermal ecosystems. Our current understanding of Movile Cave microbiology has been confined to the thermal water proximity (no more than 2 m distant), with most studies focusing on the water-floating mat, which likely acts as the primary production powerhouse in this sulfidic ecosystem. To gain more insightful information on the functioning of the sulfidic Movile Cave ecosystem, we employed a metagenomics-resolved approach to reveal the microbiome diversity, metabolic potential, and interactions and infer its roles within the food webs in the sediments beyond the sulfidic thermal waters. Results: A customized bioinformatics pipeline led to the recovery of 106 high-quality metagenome-assembled genomes from 7 cave sediment metagenomes. Assemblies’ taxonomy spanned 19 bacterial and three archaeal phyla with Acidobacteriota, Chloroflexota, Proteobacteria, Planctomycetota, Ca . Patescibacteria, Thermoproteota, Methylomirabilota , and Ca . Zixibacteria as prevalent phyla. Functional gene analyses allowed prediction of CO ₂ fixation, methanotrophy, sulfur and ammonia oxidation as possibly occurring in the explored sediments. Species Metabolic Coupling Analysis of metagenome-scale metabolic models revealed the highest competition-cooperation interactions in the sediments collected at the farthest distance from the sulfidic water. As a result of simulated metabolic interactions, autotrophs and methanotrophs were hypothesized as major donors of exchanged metabolites in the sediment communities. Cross-feeding dependencies were assumed only towards ‘currency’ molecules and inorganic compounds (O ₂ , PO ₄ ^3- , H ⁺ , Fe ²⁺ , Cu ²⁺ ) in the sediment nearby sulfidic water, whereas hydrogen sulfide and methanol are predictably traded exclusively among communities dwelling in the distant gallery. Conclusions: These findings suggest that the primary production potential of the Movile Cave expands way beyond its hydrothermal waters, enhancing our understanding of ecological interactions inside chemolithoautotrophically based subterranean ecosystems and their functioning.

In this study, we describe a sedimentary deposit situated above Ascunsă Cave (SW Romania) that should be in depositional connection with coeval stalagmites from the cave. We excavated a 2.5 m deep soil profile and took contiguous bulk samples every 5 cm. Soil samples were analyzed for clay mineralogy, grain size, chemical composition, magnetic susceptibility, and stable carbon isotopes. Radiocarbon dating revealed that the soil is of Holocene age, and presents a depositional hiatus between 5.4 and 2.3 thousand years before the present. Due to the open system behavior of soils and mobility of organic matter, a few hundred years of uncertainty should be considered for the duration of this hiatus. The fine fraction is dominated by silt, while the clay mineralogical association is made of illite, chlorite, kaolinite, vermiculite, and illite–vermiculite and illite–chlorite mixed layered minerals. The sediment source of this soil is represented by a nearby mélange complex, as well as by an underlying terra rossa-type soil. As this latter type of soil is widespread in our study area, we performed luminescence dating on two samples from a representative location close to our site and it appears that this type of Mediterranean soil was formed during the Last Interglacial period. δ¹³C variability in soil organic matter and a stalagmite from the cave are comparing well, and could help future studies identify modifications in isotopic fractionation processes within the cave.

Over the last decades, analysis of the stable isotopes of C and N has been extensively used for palaeodietary reconstruction of the MIS 3 fauna from the Romanian Carpathians. The faunal assemblages from the majority of the well-known cave sites from this region are dominated by bears, thus leaving fewer opportunities for stable isotopic analyses on other taxa. Muierilor Cave (Southern Carpathians), however, is an exception and has sedimentary deposits that yielded a taxonomically rich Late Pleistocene palaeofauna including red deer, cave bears, cave lions, wolves and hyaenas. δ15N and δ13C values derived from the bone collagen of 40 mammal individuals from this cave indicated a clear delimitation of dietary behaviors among studied faunas. In addition, red deer and carnivores from the same bone assemblages display δ15N and δ13C values similar to those of the Central and Western European MIS 3 populations. The richness of the large mammal fauna and the large sample size allowed for a better understanding of the palaeoecological relationships among these species. This study demonstrates the usefulness of δ15N and δ13C analysis in sites with a complex fossil faunal context and shows a good agreement between the newly obtained data and those reported for other European Late Pleistocene large mammals.