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The study of spring- and subterranean-associated microsnail species in the Appalachian karst region has focused disproportionately on the northern Appalachian Valley and Ridge (AVR), leaving many areas in the southern Appalachians unexplored. Consequently, biological inventories of subterranean habitats have been initiated in the southern AVR, particularly in the state of Tennessee. In 2013 and 2018, several previously unknown populations of a microsnail species were discovered from caves in eastern Tennessee. Through both morphological and molecular analysis, we identified these populations as the Blue Ridge Springsnail, Fontigens orolibas. These newly discovered populations represent a significant range extension of F. orolibas. As such, we reassess the conservation status of F. orolibas under NatureServe criteria and emphasize the need for further sampling efforts in the southern AVR for microsnails.

The North American endemic cave beetle genus Pseudanophthalmus is exceptionally diverse, with >150 described taxa in karst regions of the eastern United States. Eighty-seven per cent of taxa, however, are at risk of extinction due to small, restricted distributions, low abundance, and several potential anthropogenic threats to their habitats. Six species in Tennessee are exceedingly rare and are candidates for listing under the U.S. Endangered Species Act: Coleman Cave Beetle (P. colemanensis), Fowler's Cave Beetle (P. fowlerae), Inquirer Cave Beetle (P. inquisitor), Bakers Station Cave Beetle (P. insularis), Noblett's Cave Beetle (P. paulus), and Soothsayer Cave Beetle (P. tiresias). Each species is an extreme short-range endemic. Four species have not been observed in several decades, and two species (P. insularis and P. paulus) are considered possibly extinct. We searched 57 caves in 15 counties in Tennessee, including eight of the nine historical localities of the six Pseudanophthalmus species between July 2013 and March 2016 to determine if populations were still extant, to search for new populations, and to estimate relative abundance. We confirmed the continued existence of all six species, including P. fowlerae, P. insularis, P. paulus, and P. tiresias, which had not been observed in 52, 60, 50, and 42 years, respectively. We also discovered five new populations in total, one for each species except for P. paulus. Although U.S. Fish and Wildlife Service ruled that all six species do not warrant federal listing, all species continue to have restricted ranges and remain at an elevated risk of extinction.

A new species of cave snail (Littorinimorpha: Cochliopidae) in the genus Antrorbis is described from the dark zone of two caves in the Appalachian Valley and Ridge province in eastern Tennessee, United States. The Tennessee Cavesnail, Antrorbis tennesseensis Perez, Shoobs, Gladstone, & Niemiller, sp. nov. is distinguished from its only known congener, Antrorbis breweri, by the absence of raised tubercles on its finely spirally striate protoconch, and its unique radular formula. Moreover, A. tennesseensis is genetically distinct from A. breweri based on substantial divergence at the mitochondrial CO1 locus. This is the first cavesnail to be described from the Appalachian Valley and Ridge (AVR) physiographic province in the state of Tennessee, which previously represented a substantial gap in the distribution of stygobitic (i.e., aquatic, subterranean-obligate) gastropods.

The discovery of troglobionts in lava tube caves on the Hawaiian Islands archipelago altered our understanding of how species may evolve and permanently inhabit subterranean environments. To date, 74 troglobionts are reported from the islands with 44 known from the youngest (Big Island). Previous lava tube cave surveys on the Big Island have focused on the wetter, eastern side of the island and in lava tubes at higher elevations. Along with members of Cave Conservancy of Hawai’i, we surveyed 24 lava tube sections on the drier, western side of the Big Island, primarily in Hawaiian Ocean View Estates in the Ka’ū District. Surveys occurred during late November and early December of 2015–2017. Species present in each cave were documented by observation and limited collections of specimens for identification purposes. Significant species were discovered in these caves, representing new locations for rare and limited species previously documented from different regions on the island, and/or new, undescribed species from significant lineages that are federally listed on other islands. The most significant species observed included a reduviid thread-legged bug (Nesidiolestes sp.), a terrestrial amphipod (Spelaeorchestia sp.), a microvellid (Cavaticovelia sp.), and a new cave-adapted planthopper (Oliarus sp.). While identification of the collected specimens is ongoing, documenting these significant species from lava tube caves in Ka’ū District illustrates the need for continued bioinventory work in this area.

Several Leptodirini beetles (Leiodidae) are known to dwell in hygropetric habitats where films of water run down the cave walls, but observations of their behaviour are lacking. The ultra- specialised hygropetricolous beetles belonging to the genera Cansiliella and Hadesia are biogeographically and phylogenetically unrelated leptodirines. As the species of the former genus are known to be associated with the moonmilk deposits our study aimed to obtain data on their foraging behaviour, as well as to compare the feeding strategies of both genera. In situ monitoring of C. servadeii from the cave Grotta della Foos (Italy) and H. asamo from Bravenik Cave (Bosnia and Herzegovina), was complemented by video recordings to ensure accurate results. Mouthparts and tarsi of both species were examined using scanning electron microscopy and compared with H. weiratheri from Montenegro to evaluate potential morphological adaptations to the hygropetricolous ecological niches. The three species had significantly different mouthpart morphologies, likely due to differences in semi-aquatic feeding strategies and overall ecology. A series of new observations on site movement and feeding behaviour are presented, compared and discussed. Key words: Coleoptera, ecology, behaviour, moonmilk, cave hygropetric. Primerjava jamskih higropetricnih hroscev in njihovegaprehranjevanja; Cansiliella servadeii in Hadesia asamo (Coleoptera, Leiodidae, Cholevinae, Leptodirini) Stevilni hrosci iz poddružine Leptodirinae (Leiodidae) so prilagojeni na življenje v posebnem habitatu »jamskem higropetriku «, t.j. tankem sloju tekoce vode, ki tece po sigi. Zaenkrat je njihovo vedenje se precejsnja neznanka. Visoko specializirani higropetricni vrsti Cansiliella servadeii in Hadesia asamo sta geografsko loceni in filogenetsko nesorodni. Ker je prva ocitno vezana na depozite mehke sige (t.i. jamsko mleko), smo želeli raziskati iskanje hrane pri tej vrsti ter primerjati strategijo hranjenja obeh vrst. Da bi povecali zanesljivost rezultatov, smo poleg in situ opazovanja C. servadeii iz jame Grotta della Foos (Italija) in H. asamo iz jame Bravenik (Bosna in Hercegovina), njuno vedenje tudi posneli. Z vrsticnim elektronskim mikroskopom smo raziskali obustne okoncine in stopalca obeh vrst in jih primerjali s H. weiratheri iz Crne gore, da bi preverili potencialne morfoloske prilagoditve na specificno ekolosko niso. Bistvene razlike v zgradbi ustnega aparata so najverjetneje posledica razlik v strategiji hranjenja, kot tudi razlicne splosne ekologije omenjenih vrst. V prispevku predstavljamo, primerjamo in razpravljamo o novih opažanjih povezanih z gibanjem in prehranjevanjem higropetricnih hroscev. Kljucne besede: Coleoptera, ekologija, vedenje, jamsko mleko, jamski higropetrik.

Enhanced monsoon duration and soil acidification from acid rain are expected to impact the distribution of microbial communities in surface and subsurface environments, although these impacts are poorly understood for most systems. In central China, soluble carbonate bedrock forms extensive karst landscapes. Current predictions are that the amount of monsoonal precipitation and acid rainfall in central China will increase, which is expected to lead to changes in the pH balance of karst ecosystems. To evaluate the role of pH, total organic carbon, and other geochemical parameters (e.g., Ca2+, Mg2+, NH4+, NOx, SO42-) in shaping bacterial communities within a single karst system in central China, samples were collected from the thin surface soils overlying Heshang Cave, cave sediments, and weathered cave passage rocks from the entrance, twilight, and dark zones, as well as from epikarstic drip waters inside the cave. Illumina sequencing of 16S rRNA genes and multivariate statistical analyses revealed that each tested community was distinct and the community variability was significantly correlated with pH, total organic carbon, and potassium concentrations. Specifically, surface soils were dominated by Acidobacteria, Verrucomicrobia and Planctomycetes, and diversity significantly decreased with acidic pH values. Nitrospirae, Gemmatimonadetes, Firmicutes, and Chloroflexi were unique to cave sediments, while Actinobacteria and Proteobacteria dominated weathered rocks and drip waters, respectively. The results reveal important implications regarding the effects of acidification on bacterial communities in karst areas, and on the control of pH in shaping bacterial communities throughout a karst system. Increased water flux into and through karst habitats due to monsoonal precipitation may result in deeper penetration of acidic solutions into karst and shift the bacterial communities inside the cave in the future.

The troglobitic beetle, Cansiliella servadeii (Leptodirini), has specialized mouthparts modified for browsing and feeding under percolating water on moonmilk, a speleothem formation in Grotta della Foos, Italy. Results from analyses of stable isotopes of carbon and nitrogen suggest thatacquires and assimilates dissolved allochthonous organic carbon, inorganic nitrogen, and possibly phosphorus and other nutrients from the microbial fauna associated with moonmilk.

Chromophoric dissolved organic matter (CDOM) was examined using fluorescence and absorbance spectra from sulfidic cave and thermal and non-thermal surface-discharging spring waters. Many of the sites have a limited allochthonous supply of organic matter (OM) and contain ecosystems that are dependent on chemolithoautotrophic microbial communities. Water-extracted OM from microbial mats at the sites had fluorescence signatures consistent with the fluorescent amino acids. Based on fluorescence-derived indices and absorbance spectral characteristics, the origin of the cave and spring CDOM appeared to be from microbially-derived material, and the degree of OM humification was low. Little of the CDOM pool was represented by terrestrial humic fluorescence signatures, which are typically observed in surface waters, as well as soil and sediment porewaters. Comparison of the cave and spring waters with a wide array of reference humic substances and OM from other environments showed a continuum of spectral properties constrained by origin and degree of humification.

This study expands the phylogenetic diversity of Epsilonproteobacteria using the 16S rRNA gene framework. Of the 73 lineages defined by sequence similarities at or greater than 99%, most were found at only one site. In contrast, eight lineages were retrieved from sites spanning geographic distances from 1,000 to >10,000 km.

Summary Lower Kane Cave, Wyoming (USA), has hydrogen sulfide-bearing springs that discharge into the cave passage. The springs and cave stream harbour white filamentous microbial mats dominated by Epsilonproteobacteria. Recently, novel 16S rRNA gene sequences from the phylum Acidobacteria, sub- group 7, were found in these cave mats. Although Acidobacteria are ubiquitously distributed in many terrestrial and marine habitats, little is known about their ecophysiology. To investigate this group in Lower Kane Cave in more detail, a full-cycle rRNA approach was applied based on 16S and 23S rRNA gene clone libraries and the application of novel probes for fluorescence in situ hybridization. The 16S and 23S rRNA gene clone libraries yielded seven and six novel acidobacterial operational taxonomic units (OTUs) respectively. The majority of the OTUs were affiliated with subgroups 7 and 8. One OTU was affiliated with subgroup 6, and one OTU could not be assigned to any of the present acidobacterial subgroups. Fluorescence in situ hybridization distinguished two morphologically distinct, rod- shaped cells of the acidobacterial subgroups 7 and 8. Although the ecophysiology of Acidobacteria from Lower Kane Cave will not be fully resolved until cultures are obtained, acidobacterial cells were always associated with the potentially chemoli- thoautotrophic epsilon- or gammaproteobacterial filaments, suggesting perhaps a lifestyle based on heterotrophy or chemoorganotrophy.

Acidic biofilms present on cave walls in the sulfidic region of the Frasassi Gorge, Italy, were investigated to determine their microbial composition and their potential role in cave formation and ecosystem functioning. All biofilm samples examined had pH values < 1.0. Scanning electron microscopy of the biofilms revealed the presence of various filaments and rods associated in large clusters with mineral crystals. Qualitative energy-dispersive x-ray analysis was used to determine that the crystals present on the cave walls, associated with the microbial biofilm, were composed of calcium and barium sulfate. Ribosomal RNA-based methods to determine the microbial composition of these biofilms revealed the presence of at least two strains of potential acidophilic, sulfur-oxidizing bacteria, belonging to the genera Thiobacillus and Sulfobacillus. An acid-producing strain of Thiobacillus sp. also was obtained in pure culture. Stable isotope ratio analysis of carbon and nitrogen showed that the wall biofilms...

The microbial diversity of moonmilk, a hydrated calcium carbonate speleothem, was evaluated from two Italian caves to provide context for the food web of highly-specialized troglobitic beetles, Cansiliella spp. (Leptodirinae), with distinctive carbon and nitrogen isotope values indicative of a novel food source. The moonmilk and associated percolating waters had low to no extractable chlorophyll, with an average organic C:N ratio of 9, indicating limited allochthonous input and a significant contribution from microbial biomass. The biomass from moonmilk was estimated to be ~10 4 micro- and meiofaunal individuals per m 2 and ~10 7 microbial cells/ml. Proteobacteria dominated the 16S rRNA gene sequences retrieved from the moonmilk from both caves. The distribution of other proteobacterial classes and phyla in the moonmilk were statistically similar to each other, even though the two caves are geographically separated from each other. Comparing the moonmilk gene sequences to sequences from previously described environmental clones or cultured strains revealed the uniqueness of the moonmilk habitat, as ~15% of all of the moonmilk sequences were more closely related to each other than to sequences retrieved from any other habitat. However, comparative analyses confirmed that as much as ~34% of the clones sequences were also closely related to environmental clones and cultured strains derived from soil and freshwater habitats, which is likely due to the fact that the putative inoculation source for the moonmilk bacterial communities is from overlying soil and percolating fluids from the surface. Prior to our studies of Cansiliella spp., moonmilk has not been considered a food source for cave animals. Our findings provide unique insight into moonmilk microbial diversity that could reveal the underpinnings of the moonmilk carbon and nitrogen cycle that influences the isotopic composition and the morphological adaptations of the troglobitic beetles associated with the moonmilk. calcium carbonate; microorganisms; food web; nitrogen cycling; beetles

INTRODUCTION Whilst chemolithoautotrophic micro-organisms are found in nearly every environment on Earth, they are more abundant in dark habitats where competition by photosynthetic organisms is eliminated. Caves, particularly, represent dark but accessible subsurface habitats where the importance of microbial chemolithoautotrophy to biogeochemical and geological processes can be examined directly. At Lower Kane Cave, WY, USA, hydrogen sulfide-rich springs provide a rich energy source for chemolithoautotrophic micro-organisms, supporting a surprisingly complex consortium of micro-organisms, dominated by sulfur-oxidizing bacteria. Several evolutionary lineages within the class ‘ Epsilonproteobacteria ’ dominate the biovolume of subaqueous microbial mats, and these microbes support the cave ecosystem through chemolithoautotrophic carbon fixation. The anaerobic interior of the cave microbial mats is a habitat for anaerobic metabolic guilds, dominated by sulfate-reducing and -fermenting bacteria. Biological controls of speleogenesis had not been considered previously and it was found that cycling of carbon and sulfur through the different microbial groups directly affects sulfuric acid speleogenesis and accelerates limestone dissolution. This new recognition of the contribution of microbial processes to geological processes provides a better understanding of the causal factors for porosity development in sulfidic groundwater systems. Karst landscapes form where soluble carbonate rocks dissolve by chemical solution (karstification), resulting in numerous geomorphic features, including caves and subterranean-conduit drainage systems (e.g. White, 1988; Ford & Williams, 1989). This has traditionally been viewed as an abiotic, chemical process that occurs near the water table, with biologically produced CO 2 as the principal reactive component.

The formation of natural caves (speleogenesis) is due to any number of processes that result in the hollowing out of rock, including dissolution, mechanical weathering, volcanic activity, or even the melting of glacial ice. Caves are classified based on the solid rock that they developed within, the proximity to the groundwater table (e.g., above, at, or below it), the speleogenetic history of a feature, and the overall passage morphology and organization (e.g., cave length, passage shape, passage arrangement, passage levels) (Fig. 10.1). Caves are one type of feature that characterizes a karst landscape, which develops in soluble rocks (e.g., limestone, dolomite, gypsum, halite) that roughly coincides with the global distribution of carbonate sedimentary rocks of all geologic ages (e.g., Ford and Williams 2007). Although karst comprises ∼15–20% of the Earth’s ice-free land surface, karst caves are not interconnected, not within the same hydrological drainage basin and definitely not across different drainage basins.

Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, where microbial mats dominated by filamentous morphotypes are found. The full-cycle rRNA approach, including 16S rRNA gene retrieval and fluorescence in situ hybridization (FISH), was used to identify these filaments. The majority of the obtained 16S rRNA gene clones from the mats were affiliated with the “ Epsilonproteobacteria ” and formed two distinct clusters, designated LKC group I and LKC group II, within this class. Group I was closely related to uncultured environmental clones from petroleum-contaminated groundwater, sulfidic springs, and sulfidic caves (97 to 99% sequence similarity), while group II formed a novel clade moderately related to deep-sea hydrothermal vent symbionts (90 to 94% sequence similarity). FISH with newly designed probes for both groups specifically stained filamentous bacteria within the mats. FISH-based quantification of the two filament groups in six different microbial mat samples from Lower Kane Cave showed that LKC group II dominated five of the six mat communities. This study further expands our perceptions of the diversity and geographic distribution of “ Epsilonproteobacteria ” in extreme environments and demonstrates their biogeochemical importance in subterranean ecosystems.

The sulfuric acid speleogenesis (SAS) model was introduced in the early 1970s from observations of Lower Kane Cave, Wyoming, and was proposed as a cave-enlargement process due to primarily H 2 S autoxidation to sulfuric acid and subaerial replacement of carbonate by gypsum. Here we present a reexamination of the SAS type locality in which we make use of uniquely applied geochemical and microbiological methods. Little H 2 S escapes to the cave atmosphere, or is lost by abiotic autoxidation, and instead the primary H 2 S loss mechanism is by subaqueous sulfur-oxidizing bacterial communities that consume H 2 S. Filamentous “ Epsilonproteobacteria ” and Gammaproteobacteria , characterized by fluorescence in situ hybridization, colonize carbonate surfaces and generate sulfuric acid as a metabolic byproduct. The bacteria focus carbonate dissolution by locally depressing pH, compared to bulk cave waters near equilibrium or slightly supersaturated with calcite. These findings show that SAS occurs in subaqueous environments and potentially at much greater phreatic depths in carbonate aquifers, thereby offering new insights into the microbial roles in subsurface karstification.