The microbially mediated replacement of sulfate-bearing evaporites by authigenic carbonate and native sulfur under anoxic conditions is poorly understood. Sulfur-bearing carbonates from the Monte Palco ridge (Sicily) replacing Messinian gypsum were therefore studied to better characterize the involved microorganisms. The lack of (1) sedimentary bedding, (2) lamination, and (3) significant water-column-derived lipid biomarkers in the secondary carbonates implies replacement after gypsum deposition (epigenesis). Allochthonous clasts from the older Calcare di Base and the younger Trubi Formation within these carbonates further evidence epigenetic formation. The sulfur-bearing carbonates are significantly ¹³C-depleted (δ¹³C as low as –51%), identifying methane as a major carbon source. The ¹⁸O-enrichment of the carbonates (δ¹⁸O as high as 5.4%) probably reflects precipitation from ¹⁸O-enriched fluids transported along adjacent faults or precipitation in a closed system with very little water. Native sulfur with variable ³⁴S-enrichment (δ³⁴S as high as 18.9%), a relatively small maximum offset (12.3%) between the sulfate source (gypsum) and native sulfur, and high δ³⁴S values of carbonate-associated sulfate (as high as 61.1%) suggest a high conversion to native sulfur in a (semi-)closed system, with insignificant sulfate removal. Anaerobic methanotrophic archaea (ANME) apparently affiliated with the ANME-1 clade mediated secondary mineral formation as evidenced by the biomarker inventory, which contains abundant ¹³C-depleted isoprenoids including sn3-hydroxyarchaeol as the sole hydroxyarchaeol isomer and glycerol dibiphytanyl glycerol tetraethers (GDGTs). A series of various, tentatively identified ¹³C-depleted non-isoprenoidal dialkyl glycerol diethers (DAGEs), 10me-C₁₆ fatty acid, hydroxy C₁₆ fatty acids, and cyclopropyl-C_{17:0ω7,8 fatty acid agree with sulfate-reducing bacteria participating in the anaerobic oxidation of methane. Specific conditions during gypsum replacement, unlike those at marine methane seeps, are reflected by the occurrence of ¹³C-depleted lipids such as lycopane, 9me-C17 fatty acid, and novel DAGEs. As a response to a confined environment probably characterized by high sulfate concentrations, sulfidic conditions, and elevated salinity, ANMEs and sulfate-reducing bacteria apparently adapted their membrane compositions to cope with such stressors.}

Lehman Caves is an extensively decorated high desert cave that represents one of the main tourist attractions in Great Basin National Park, Nevada. Although traditionally considered a water table cave, recent studies identified abundant speleogenetic features consistent with a hypogenic and, potentially, sulfuric acid origin. Here, we characterized white mineral deposits in the Gypsum Annex (GA) passage to determine whether these secondary deposits represent biogenic minerals formed during sulfuric acid corrosion and explored microbial communities associated with these and other mineral deposits throughout the cave. Powder X-ray diffraction (pXRD), scanning electron microscopy with electron dispersive spectroscopy (SEM-EDS), and electron microprobe analyses (EPMA) showed that, while most white mineral deposits from the GA contain gypsum, they also contain abundant calcite, silica, and other phases. Gypsum and carbonate-associated sulfate isotopic values of these deposits are variable, with δ³⁴S_V-CDT between +9.7% and +26.1%, and do not reflect depleted values typically associated with replacement gypsum formed during sulfuric acid speleogenesis. Petrographic observations show that the sulfates likely co-precipitated with carbonate and SiO₂ phases. Taken together, these data suggest that the deposits resulted from later-stage meteoric events and not during an initial episode of sulfuric acid speleogenesis. Most sedimentary and mineral deposits in Lehman Caves have very low microbial biomass, with the exception of select areas along the main tour route that have been impacted by tourist traffic. High-throughput 16S rRNA gene amplicon sequencing showed that microbial communities in GA sediments are distinct from those in other parts of the cave. The microbial communities that inhabit these oligotrophic secondary mineral deposits include OTUs related to known ammonia-oxidizing Nitrosococcales and Thaumarchaeota, as well as common soil taxa such as Acidobacteriota and Proteobacteria. This study reveals microbial and mineralogical diversity in a previously understudied cave and expands our understanding of the geomicrobiology of desert hypogene cave systems.