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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.

Lucinid bivalves harbor environmentally acquired, chemosynthetic, gammaproteobacterial gill endosymbionts. Lucinid gill microbiomes, which may contain other gammaproteobacterial and/or spirochete taxa, remain under-sampled. To understand inter-host variability of the lucinid gill microbiome, specifically in the bacterial communities, we analyzed the microbiome content of Stewartia floridana collected from Florida. Sampled gills contained a monospecific gammaproteobacterial endosymbiont expressing lithoautotrophic, mixotrophic, diazotrophic and C1 compound oxidation-related functions previously characterized in similar lucinid species. Another low-abundance Spirochaeta-like species in ∼72% of the sampled gills was most closely related to Spirochaeta-like species in another lucinid Phacoides pectinatus and formed a clade with known marine Spirochaeta symbionts. The spirochete expressed genes were involved in heterotrophy and the transport of sugars, amino acids, peptides and other substrates. Few muscular and neurofilament genes from the host and none from the gammaproteobacterial and spirochete symbionts were differentially expressed among quadrats predominantly covered with seagrass species or 80% bare sand. Our results suggest that spirochetes are facultatively associated with S. floridana, with potential scavenging and nutrient cycling roles. Expressed stress- and defense-related functions in the host and symbionts also suggest species–species communications, which highlight the need for further study of the interactions among lucinid hosts, their microbiomes and their environment.

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.

The preservation of vertebrate fossils requires transformation of the original bioapatite mineral matrix, predominantly hydroxylapatite, during diagenesis into a thermodynamically more stable phase. Fossil bone geochemistry and structure provides a wealth of information on the conditions present during diagenesis, including site redox and pH, and about sedimentation rates during burial. The Gray Fossil Site preserves Late Neogene lacustrine deposits that serve as one of a few records of North American paleoclimate during a critical period of the Cenozoic. Despite having > 40 vertebrate species and diverse plant assemblages, none of the prior studies used bone composition to understand what paleoenvironmental and diagenetic conditions must have been like to facilitate bone preservation and fossilization. Alligator sp. phalanges were examined using multiple geochemical tools, including Fourier-transform infrared spectroscopy, X-ray diffraction, and electron microprobe analyses. Evidence of bioerosion within thin cortical and isolated trabecular regions of bone suggests biotic utilization of organics (collagen) and/or the mineral(s) following deposition. Chemically zoned and laminated cortical Alligator bone is interpreted as preserved lines of arrested growth that developed during life, and supports previous interpretations of a regional seasonal climate. Lastly, the Alligator bones are mineralogically heterogeneous as fluorinated and iron-containing apatite phases. Compared to modern bone, iron concentrations were high, at up to 3 wt%. The uptake of Fe by the Alligator bones examined in this study suggests that fluids present during diagenesis that led to bone preservation within the lacustrine sediment at the Gray Fossil Site were likely acidic, anoxic, and reducing.

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.

Symbiont diversity and host/symbiont functions have been comprehensively profiled for only a few lucinid species. In this work, unprecedented thioautotrophic gill endosymbiont taxonomic diversity was characterized within a Ctena orbiculata population associated with both seagrass- and alga-covered sediments. Endosymbiont metabolisms included known chemosynthetic functions and an additional conserved, previously uncharacterized C1 oxidation pathway. Lucinid-symbiont associations were not species specific because this C. orbiculata population hosted multiple endosymbiont strains and species, and other sympatric lucinid species shared overlapping symbiont 16S rRNA gene diversity profiles with C. orbiculata. Our results suggest that lucinid-symbiont association patterns within some host species could be more taxonomically diverse than previously thought. As such, this study highlights the importance of holistic analyses, at the population, community, and even ecosystem levels, in understanding host-microbe association patterns., Seagrass-dwelling members of the bivalve family Lucinidae harbor environmentally acquired gill endosymbionts. According to previous studies, lucinid symbionts potentially represent multiple strains from a single thioautotrophic gammaproteobacterium species. This study utilized genomic- and transcriptomic-level data to resolve symbiont taxonomic, genetic, and functional diversity from Ctena orbiculata endosymbiont populations inhabiting carbonate-rich sediment at Sugarloaf Key, FL (USA). The sediment had mixed seagrass and calcareous green alga coverage and also was colonized by at least five other lucinid species. Four coexisting, thioautotrophic endosymbiont operational taxonomic units (OTUs), likely representing four strains from two different bacterial species, were identified from C. orbiculata. Three of these OTUs also occurred at high relative abundances in the other sympatric lucinid species. Interspecies genetic differences averaged about 5% lower at both pairwise average nucleotide identity and amino acid identity than interstrain differences. Despite these genetic differences, C. orbiculata endosymbionts shared a high number of metabolic functions, including highly expressed thioautotrophy-related genes and a moderately to weakly expressed conserved one-carbon (C1) oxidation gene cluster previously undescribed in lucinid symbionts. Few symbiont- and host-related genes, including those encoding symbiotic sulfurtransferase, host respiratory functions, and host sulfide oxidation functions, were differentially expressed between seagrass- and alga-covered sediment locations. In contrast to previous studies, the identification of multiple endosymbiont taxa within and across C. orbiculata individuals, which were also shared with other sympatric lucinid species, suggests that neither host nor endosymbiont displays strict taxonomic specificity. This necessitates further investigations into the nature and extent of specificity of lucinid hosts and their symbionts. IMPORTANCE Symbiont diversity and host/symbiont functions have been comprehensively profiled for only a few lucinid species. In this work, unprecedented thioautotrophic gill endosymbiont taxonomic diversity was characterized within a Ctena orbiculata population associated with both seagrass- and alga-covered sediments. Endosymbiont metabolisms included known chemosynthetic functions and an additional conserved, previously uncharacterized C1 oxidation pathway. Lucinid-symbiont associations were not species specific because this C. orbiculata population hosted multiple endosymbiont strains and species, and other sympatric lucinid species shared overlapping symbiont 16S rRNA gene diversity profiles with C. orbiculata. Our results suggest that lucinid-symbiont association patterns within some host species could be more taxonomically diverse than previously thought. As such, this study highlights the importance of holistic analyses, at the population, community, and even ecosystem levels, in understanding host-microbe association patterns.

One of the most challenging problems in paleobiology is determining how bone transforms from a living tissue into a fossil. The geologic record is replete with vertebrate fossils preserved from a range of depositional environments, including wetland systems. However, thermodynamic models suggest that bone (modeled as hydroxylapatite) is generally unstable in a range of varying geochemical conditions and should readily dissolve if it does not alter to a more thermodynamically stable phase, such as a fluorine-enriched apatite. Here, we assess diagenesis of alligator bone from fleshed, articulated skeletons buried in wetland soils and from de-fleshed bones in experimental mesocosms with and without microbial colonization. When microbial colonization of bone was inhibited, bioapatite recrystallization to a more stable apatite phase occurred after one month of burial. Ca-Fe-phosphate phases in bone developed after several months to years due to ion substitutions from the protonation of the hydroxyl ion. These rapid changes demonstrate a continuum of structural and bonding transformations to bone that have not been observed previously. When bones were directly in contact with sediment and microbial cells, rapid bioerosion and compositional alteration occurred after one week, but slowed after one month because biofilms reduced exposed surfaces and subsequent bioapatite lattice substitutions. Microbial contributions are likely essential in forming stable apatite phases during early diagenesis and for enabling bone preservation and fossilization.

In the central and eastern United States, cavefishes have been known historically only from the Interior Low Plateau and Ozarks karst regions. Previously, cavefishes were unknown from the Appalachians karst region, which extends from southeastern New York southwestward into eastern Tennessee, northwestern Georgia, and northeastern Alabama. Here we report the discovery of a new population of the amblyopsid cavefish Typhlichthys subterraneus Girard, 1859 from a cave in Catoosa County, Georgia, that significantly extends the known distribution of the species. The cave is located in the Appalachian Valley and Ridge physiographic province and Appalachians karst region, and represents the first definitive report of a stygobiotic fish from the Appalachians karst region. Genetic analyses of one mitochondrial and one nuclear locus from the cavefish indicate this population is closely allied with populations that occur along the western margins of Lookout and Fox mountains in Dade County, Georgia, and populations to the northwest in southern Marion County, Tennessee. It is likely that these populations are also related to those from Wills Valley, DeKalb County, Alabama. The distribution of this new population of T. subterraneus and its close allies pre-dates the emergence of a Tennessee-Coosa River drainage divide in the Pliocene. The potential exists to discover additional populations in caves within the Appalachians karst region in Catoosa County and northward into Hamilton County, Tennessee.

The hydrothermal fluid chemistry at El Tatio Geyser Field (ETGF) in northern Chile suggests that biogenic CO2–CH4 cycling may play an important role in water chemistry, and relatively low sulfate (0.6–1 mM) and high molecular hydrogen (H2) concentrations (67–363 nM) suggest that methanogenic Archaea are present in ETGF microbial mats. In this study, δ13C analysis of dissolved inorganic carbon and methane was not indicative of biogenic methane production (δ13CCH4 values ranging from −15‰ to −5.3‰); however, methanogenic Archaea were successfully cultured from each of the hydrothermal sites sampled. Sanger sequencing using universal Archaea primers identified putative methanogenic orders with varying metabolic capabilities, including Methanobacteriales, Methanomicrobiales and Methanosarcinales.

The prevailing paradigm in subterranean ecology is that below-ground food webs are simple, limited to one or two trophic levels, and composed of generalist species because of spatio-temporally patchy food resources and pervasive energy limitation. This paradigm is based on relatively few studies of easily accessible, air-filled caves. However, in some subterranean ecosystems, chemolithoautotrophy can subsidize or replace surface-based allochthonous inputs of photosynthetically derived organic matter (OM) as a basal food resource and promote niche specialization and evolution of higher trophic levels. Consequently, the current subterranean trophic paradigm fails to account for variation in resources, trophic specialization, and food chain length in some subterranean ecosystems. We reevaluated the subterranean food web paradigm by examining spatial variation in the isotopic composition of basal food resources and consumers, food web structure, stygobiont species diversity, and chromophoric organic matter (CDOM), across a geochemical gradient in a large and complex groundwater system, the Edwards Aquifer in Central Texas (USA). Mean δ13C values of stygobiont communities become increasingly more negative along the gradient of photosynthetic OM sources near the aquifer recharge zone to chemolithoautotrophic OM sources closer to the freshwater-saline water interface (FWSWI) between oxygenated freshwater and anoxic, sulfide-rich saline water. Stygobiont community species richness declined with increasing distance from the FWSWI. Bayesian mixing models were used to estimate the relative importance of photosynthetic OM and chemolithoautorophic OM for stygobiont communities at three biogeochemically distinct sites. The contribution of chemolithoautotrophic OM to consumers at these sites ranged between 25% and 69% of total OM utilized and comprised as much as 88% of the diet for one species. In addition, the food web adjacent to the FWSWI had greater trophic diversity when compared to the other two sites. Our results suggest that diverse OM sources and in situ, chemolithoautotrophic OM production can support complex groundwater food webs and increase species richness. Chemolithoautotrophy has been fundamental for the long-term maintenance of species diversity, trophic complexity, and community stability in this subterranean ecosystem, especially during periods of decreased photosynthetic production and groundwater recharge that have occurred over geologic time scales.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Lucinidae clams harbor gammaproteobacterial thioautotrophic gill endosymbionts that are environmentally acquired. Thioautotrophic lucinid symbionts are related to metabolically similar symbionts associated with diverse marine host taxa and fall into three distinct phylogenetic clades. Most studies on the lucinid–bacteria chemosymbiosis have been done with seagrass-dwelling hosts, whose symbionts belong to the largest phylogenetic clade. In this study, we examined the taxonomy and functional repertoire of bacterial endosymbionts at an unprecedented resolution from Phacoides pectinatus retrieved from mangrove-lined coastal sediments, which are underrepresented in chemosymbiosis studies. The P. pectinatus thioautotrophic endosymbiont expressed metabolic gene variants for thioautotrophy, respiration, and nitrogen assimilation distinct from previously characterized lucinid thioautotrophic symbionts and other marine symbionts. At least two other bacterial species with different metabolisms were also consistently identified in the P. pectinatus gill microbiome, including a Kistimonas-like species and a Spirochaeta-like species. Bacterial transcripts involved in adhesion, growth, and virulence and mixotrophy were highly expressed, as were host-related hemoglobin and lysozyme transcripts indicative of sulfide/oxygen/CO2 transport and bactericidal activity. This study suggests the potential roles of P. pectinatus and its gill microbiome species in mangrove sediment biogeochemistry and offers insights into host and microbe metabolisms in the habitat.

Changes to hydroxylapatite mineral structure, composition, and lattice configuration must occur during the transformation of fresh bone to fossil bioapatite mineral phases. A paucity of analytical methods to characterize atomic-level lattice composition and bonding arrangements in modern and fossil bone has resulted in limited knowledge of how depositional environments, and thus diagenetic conditions, may influence fossilization processes. Synchrotron-based analyses can provide insight into bone fossilization, specifically X-ray absorption near edge structure (XANES) spectroscopy at the P and Ca K-edges. To aid in the interpretation of XANES spectral data from modern bones, and from Late Neogene and Late Cretaceous fossil bones, we applied other established techniques used to characterize bone mineralogy and composition, including X-ray diffraction, Fourier transform infrared spectroscopy, electron microprobe analysis, and thermal analysis. Specific bone depositional origins and ages likely influenced the varying elemental chemistry, preservation of organic matter, and mineral content. Fossilized bones, ranging from an almost pure fluorapatite to carbonated fluorapatite mineral phases, had less preserved organic material than modern bone. The atomic-level P and Ca lattice structures of fossilized bones differed from modern bones, specifically having shifted and shortened Ca–O bonds due to Ca 2 + substitution by ionic phases, such as iron or rare earth elements, and carbonate substitution for phosphate. The modified lattice structure, regardless of bulk mineral chemistry, provides thermodynamic stability in geochemically dynamic depositional environments that may dictate bioapatite preservation over geologic time.

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.

Coastal salt marshes along the northern Gulf of Mexico shoreline received varied types and amounts of weathered oil residues after the 2010 Deepwater Horizon oil spill. At the time, predicting how marsh bacterial communities would respond and/or recover to oiling and other environmental stressors was difficult because baseline information on community composition and dynamics was generally unavailable. Here, we evaluated marsh vegetation, physicochemistry, flooding frequency, hydrocarbon chemistry, and subtidal sediment bacterial communities from 16S rRNA gene surveys at 11 sites in southern Louisiana before the oil spill and resampled the same marshes three to four times over 38 months after the spill. Calculated hydrocarbon biomarker indices indicated that oil replaced native natural organic matter (NOM) originating from Spartina alterniflora and marine phytoplankton in the marshes between May 2010 and September 2010. At all the studied marshes, the major class- and order-level shifts among the phyla Proteobacteria , Firmicutes , Bacteroidetes , and Actinobacteria occurred within these first 4 months, but another community shift occurred at the time of peak oiling in 2011. Two years later, hydrocarbon levels decreased and bacterial communities became more diverse, being dominated by Alphaproteobacteria ( Rhizobiales ), Chloroflexi ( Dehalococcoidia ), and Planctomycetes . Compositional changes through time could be explained by NOM source differences, perhaps due to vegetation changes, as well as marsh flooding and salinity excursions linked to freshwater diversions. These findings indicate that persistent hydrocarbon exposure alone did not explain long-term community shifts. IMPORTANCE Significant deterioration of coastal salt marshes in Louisiana has been linked to natural and anthropogenic stressors that can adversely affect how ecosystems function. Although microorganisms carry out and regulate most biogeochemical reactions, the diversity of bacterial communities in coastal marshes is poorly known, with limited investigation of potential changes in bacterial communities in response to various environmental stressors. The Deepwater Horizon oil spill provided an unprecedented opportunity to study the long-term effects of an oil spill on microbial systems in marshes. Compared to previous studies, the significance of our research stems from (i) a broader geographic range of studied marshes, (ii) an extended time frame of data collection that includes prespill conditions, (iii) a more accurate procedure using biomarker indices to understand oiling, and (iv) an examination of other potential stressors linked to in situ environmental changes, aside from oil exposure.

We measured the concentration of petroleum hydrocarbons in 405 wetland sediment samples immediately before the April 2010 Deepwater Horizon disaster led to their broad-scale oiling, and on nine trips afterwards. The average concentrations of alkanes and PAHs were 604 and 186 times the pre-spill baseline values, respectively. Oil was distributed with some attenuation up to 100m inland from the shoreline for alkanes, but increased for aromatics, and was not well-circumscribed by the rapid shoreline assessments (a.k.a. SCAT) of relative oiling. The concentrations of target alkanes and PAHs in June 2013 were about 1% and 5%, respectively, of the February 2011 concentrations, but remained at 3.7 and 33 times higher, respectively, than in May 2010. A recovery to baseline conditions suggests that the concentration of alkanes may be near baseline values by the end of 2015, but that it may take decades for the PAH concentrations to be that low.

Despite the tremendous importance for geologic systems, there is limited knowledge of sulfur-containing gas-phase geochemical reactions that take place at elevated temperatures, including up to 1000 K. This deficit is at least partly caused by a lack of suitable experimental techniques to monitor and quantify potential reactions. We developed a new furnace design that can heat solid samples to specific temperatures while in situ X-ray absorption spectra of the gas-phase species are collected at the sulfur K-edge at about 2400 eV for X-ray absorption near edge structure (XANES) spectroscopy. Our experimental design improves previous furnace systems developed to generate sulfur vapors for spectrometry measurements and earlier sulfur XANES spectroscopic analyses of sulfur-containing gases and vapors. The new design minimizes contamination by air, water vapor, and stainless steel. Our results demonstrate that vapors emitted from elemental sulfur heated to different temperatures are similar to previous findings, but we can now resolve long-held interpretative discrepancies for sulfur vapor speciation, some of which resulted from the misidentification of vapors due to reactions between the sulfur vapors and furnace materials. We also used the new design to quantify the sulfur vapor species produced from elemental sulfur reacted with water over a range of temperatures. At low temperatures, like 363 K (~ 90 °C), the gas consisted of different sulfur species, whereas at temperatures greater than 550 K (~ 280 °C), the gas present in the furnace was only SO 2 . These quantitative findings show promise that future reaction kinetics can be done by using our experimental spectrometry system.

The Coastal Waters Consortium (CWC) led by Louisiana Universities Marine Consortium is one of eight Gulf of Mexico Research Initiative research consortia. The CWC focuses on: oil transport and fate, chemical evolution and biological degradation, and environmental effects.The following is an overview of a portion of the research conducted within the consortium. The consortium works in a system that was impacted by the Deepwater Horizon oil disaster and additionally impacted by freshwater diversions resulting in changes in salinity, tropical storms, and hurricanes. First, we conducted model simulations assessing oil transport into the Barataria Bay estuary, which indicate that easterly winds and feeding of the anticyclonic gyre in the Louisiana Bight pushed the oil into Barataria Bay. In subtidal sediments adjacent to oiled marshes, marsh detritus from eroding marsh edges eventually became entrained in the sediment column. Biotic impacts vary. The above-ground plant biomass appears healthy at the individual sampling sites; overall the most seaward (i.e., likely oil-impacted) areas of Terrebonne and Barataria Bay have shown, via satellite data, a distinct decline in marsh vegetation coverage since 2010. Oysters appear to be affected by predation and salinity variation. Microbial diversity from marsh-edge sediments is distinct from before and after the spill, and between unoiled and oiled marshes, with lower diversity in oiled marshes; but the greatest community composition shifts are in marshes affected by the freshwater diversions. Changes in microbial diversity in the water column at the stream-side edge of oiled marshes are extensive and are related to marsh edge erosion. In contrast, oiling of marshes had no impact on ammonia oxidizer or denitrifier abundances and on soil biogeochemical process rates 2+ years post-spill. Analysis of long-term offshore phytoplankton community and hypoxia data indicate some signal of the Macondo oil, but these components of the ecosystem remain mostly influenced by the fresh water and nutrients delivered by the Mississippi River. The consortium continues to work to tease apart oil impacts, effects of salinity, natural variation, and disturbance from tropical storms and hurricanes to determine the trajectory for health of shelf waters and Louisiana's marshes.

Arsenic concentrations (450–600 μmol L−1) at the El Tatio Geyser Field in northern Chile are an order of magnitude greater than at other natural geothermal sites, making El Tatio an ideal location to investigate unique microbial diversity and metabolisms associated with the arsenic cycle in low sulfide, > 50 °C, and circumneutral pH waters. 16S rRNA gene and arsenite oxidase gene ( aio A) diversities were evaluated from biofilms and microbial mats from two geyser-discharge stream transects. Chloroflexi was the most prevalent bacterial phylum at flow distances where arsenite was converted to arsenate, corresponding to roughly 60 °C. Among aio A-like gene sequences retrieved, most had homology to whole genomes of Chloroflexus aurantiacus , but others were homologous to alphaproteobacterial and undifferentiated beta- and gammaproteobacterial groups. No D einococci , T hermus , A quificales , or C hlorobi aio A-like genes were retrieved. The functional importance of amino acid sites was evaluated from evolutionary trace analyses of all retrieved aio A genes. Fifteen conserved residue sites identified across all phylogenetic groups highlight a conserved functional core, while six divergent sites demonstrate potential differences in electron transfer modes. This research expands the known distribution and diversity of arsenite oxidation in natural geothermal settings, and provides information about the evolutionary history of microbe–arsenic interactions.

Despite the ecological and economic importance of Thalassia testudinum (turtle grass) meadows along the Caribbean and Gulf of Mexico coasts, and recognition that microbial activities are critical to plant growth and health, the bacterial diversity of these habitats has been poorly studied. Based on comparative analyses of 16S rRNA gene sequences from sediments in a T. testudinum meadow, 25 major taxonomic groups (excluding candidate divisions) were retrieved, including Alpha - Delta -, and Gamma-proteobacteria , Chloroflexi , Bacteroidetes , Acidobacteria , Spirochaetes , and Firmicutes . The distribution of bacterial groups was linked to a strongly hypoxic and sulfidic redox gradient. The diversity is potentially novel because phylogenetic affinities of sediment sequences compared to contextually annotated environmental clones from different habitats or to cultured representatives indicated approximately 41% were more closely related to each other than to sequences retrieved from these other habitats. Of all the relationships, very few (2.4%) were to cultured organisms, but 27% were to environmental clones retrieved from shallow marine shelf and coastal sediments or from mangroves, estuarine, or wetland sediments. Rare sequences were closely related to endosymbiont groups of Lucinisca nassula (Lucinidea: Bivalvia) hosts collected from the same meadow, which may indicate that the sediment is a potential reservoir for free-living symbionts. This study provides insight into the ecological and evolutionary relationships of the Thalassia –lucinid–bacteria system in tropical to sub-tropical regions.

Epsilonproteobacteria are widely distributed in marine, freshwater, and terrestrial environments, although most well-studied groups are from hydrothermal vents and the human intestinal tract. The environmental variables that control epsilonproteobacterial communities in sulfidic terrestrial environments, however, are poorly understood. Here, the environmental variables that influence epsilonproteobacterial community composition in geographically separated sulfidic caves and springs were determined by coarse and fine-scale approaches: denaturing gradient gel electrophoresis profiling of 23S rRNA PCR amplicons and clone library sequencing of the 16S-ITS-23S rRNA operon. Sequences retrieved from this study were not closely related to cultured representatives, indicating that existing culture collections do not adequately capture the diversity of terrestrial Epsilonproteobacteria. Comparisons of 16S-ITS-23S rRNA operon sequences from four sites revealed that some distant communities (> 8000 km) share closely related populations of Epsilonproteobacteria, while other sites have nearly clonal and phylogenetically distinct populations. Statistical evaluations of sequence data reveal that multiple environmental variables (e.g. temperature, pH, salinity, dissolved oxygen, and bicarbonate concentrations) influence Epsilonproteobacteria community composition. Locations with clonal populations tended to be from higher temperatures and intermediate dissolved oxygen concentrations. rRNA operon sequences outside of the 16S rRNA gene may be critical to recognizing environmental drivers of epsilonproteobacterial community composition.

Microbially induced carbonate dissolution was evaluated from sulfidic wells in the Edwards Aquifer, Texas. Filamentous biomass covers rock surfaces, with Gammaproteobacteria and Epsilonproteobacteria dominating the attached community, but novel Alphaproteobacteria dominating the planktonic community. Despite fluids being saturated with respect to calcite, experimental calcite from in situ microcosms had significantly greater mass loss when colonized. Moreover, neoformed gypsum crystals were observed on colonized surfaces where fluids were undersaturated with respect to gypsum. The results are similar to findings from shallow cave and karst environments, and highlight the underappreciated role of microbes in the modification of carbonate aquifers and reservoirs.

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.

Microbial mats in sulfidic cave streams offer unique opportunities to study redox-based biogeochemical nutrient cycles. Previous work from Lower Kane Cave, Wyoming, USA, focused on the aerobic portion of microbial mats, dominated by putative chemolithoautotrophic, sulfur-oxidizing groups within the Epsilonproteobacteria and Gammaproteobacteria. To evaluate nutrient cycling and turnover within the whole mat system, a multidisciplinary strategy was used to characterize the anaerobic portion of the mats, including application of the full-cycle rRNA approach, the most probable number method, and geochemical and isotopic analyses. Seventeen major taxonomic bacterial groups and one archaeal group were retrieved from the anaerobic portions of the mats, dominated by Deltaproteobacteria and uncultured members of the Chloroflexi phylum. A nutrient spiraling model was applied to evaluate upstream to downstream changes in microbial diversity based on carbon and sulfur nutrient concentrations. Variability in dissolved sulfide concentrations was attributed to changes in the abundance of sulfide-oxidizing microbial groups and shifts in the occurrence and abundance of sulfate-reducing microbes. Gradients in carbon and sulfur isotopic composition indicated that released and recycled byproduct compounds from upstream microbial activities were incorporated by downstream communities. On the basis of the type of available chemical energy, the variability of nutrient species in a spiraling model may explain observed differences in microbial taxonomic affiliations and metabolic functions, thereby spatially linking microbial diversity to nutrient spiraling in the cave stream ecosystem.

The abundance of As and Sb in aqueous, mineral and biological reservoirs was examined at El Tatio Geyser Field, a unique hydrothermal basin located in the Atacama Desert region of Chile. Here the concentration of total As and Sb in hydrothermal springs and discharge streams are the highest reported for a natural surface water, and the geyser basin represents a significant source of toxic elements for downstream users across Region II, Chile. The geyser waters are near neutral Na:Cl type with ∼0.45 and 0.021 mmol L −1 total As and Sb, respectively, primarily in the reduced (III) redox state at the discharge with progressive oxidation downstream. The ferric oxyhydroxides associated with the microbial mats and some mineral precipitates accumulate substantial As that was identified as arsenate by XAS analysis (>10 wt% in the mats). This As is easily mobilized by anion exchange or mild dissolution of the HFO, and the ubiquitous microbial mats represent a significant reservoir of As in this system. Antimony, in contrast, is not associated with the mineral ferric oxides or the biomats, but is substantially enriched in the silica matrix of the geyserite precipitates, up to 2 wt% as Sb 2 O 3 . Understanding the mobility and partitioning behavior of these metalloids is critical for understanding their eventual impact on regional water management.

Although ecosystems thriving in the absence of photosynthetic processes are no longer considered unique phenomena, we haveyet to understand how these ecosystems are energetically sustained via chemosynthesis. Ecosystem energetics were measuredin microbial mats from active sulfidic caves (Movile Cave, Romania; Frasassi Caves, Italy; Lower Kane Cave, Wyoming, USA; andCesspool Cave, Virginia, USA) using radiotracer techniques. We also estimated bacterial diversity using 16S rRNA sequences torelate the productivity measurements to the composition of the microbial communities. All of the microbial communities investigatedwere dominated by chemolithoautotrophic productivity, with the highest rates from Movile Cave at 281 g C/m2/yr. Heterotrophicproductivities were at least one order of magnitude less than autotrophy from all of the caves. We generated 414 new 16S rRNAgene sequences that represented 173 operational taxonomic units (OTUs) with 99% sequence similarity. Although 13% of theseOTUs were found in more than one cave, the compositions of each community were significantly different from each other (P≤0.001).Autotrophic productivity was positively correlated with overall species richness and with the number of bacterial OTUs affiliated withthe Epsilonproteobacteria, a group known for sulfur cycling and chemolithoautotrophy. Higher rates of autotrophy were also stronglypositively correlated to available metabolic energy sources, and specifically to dissolved sulfide concentrations. The relationship ofautotrophic productivity and heterotrophic cycling rates to bacterial species richness can significantly impact the diversity of highertrophic levels in chemolithoautotrophically-based cave ecosystems, with the systems possessing the highest productivity supportingabundant and diverse macro-invertebrate communities.