Gill microbiome structure and function in the chemosymbiotic coastal lucinid Stewartia floridana.

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.

Correction: Taxonomic and functional heterogeneity of the gill microbiome in a symbiotic coastal mangrove lucinid species.

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

Extensive Thioautotrophic Gill Endosymbiont Diversity within a Single Ctena orbiculata (Bivalvia: Lucinidae) Population and Implications for Defining Host-Symbiont Specificity and Species Recognition.

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.

Taxonomic and functional heterogeneity of the gill microbiome in a symbiotic coastal mangrove lucinid species.

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.

Salt Marsh Bacterial Communities before and after the Deepwater Horizon Oil Spill.

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.

Chemolithoautotrophy supports macroinvertebrate food webs and affects diversity and stability in groundwater communities.

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.

Distribution and recovery trajectory of Macondo (Mississippi Canyon 252) oil in Louisiana coastal wetlands.

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.

Regime shift in sandy beach microbial communities following Deepwater Horizon oil spill remediation efforts.

Sandy beaches support a wide variety of underappreciated biodiversity that is critical to coastal ecosystems. Prior to the 2010 Deepwater Horizon oil spill, the diversity and function of supratidal beach sediment microbial communities along Gulf of Mexico coastlines were not well understood. As such, it was unclear if microbial community compositional changes would occur following exposure to beached oil, if indigenous communities could biodegrade oil, or how cleanup efforts, such as sand washing and sediment redistribution, would impact microbial ecosystem resiliency. Transects perpendicular to the shoreline were sampled from public beaches on Grand Isle, Louisiana, and Dauphin Island, Alabama, over one year. Prior to oil coming onshore, elevated levels of bacteria associated with fecal contamination were detected (e.g., Enterobacteriales and Campylobacterales). Over time, significant shifts within major phyla were identified (e.g., Proteobacteria, Firmicutes, Actinobacteria) and fecal indicator groups were replaced by taxa affiliated with open-ocean and marine systems (e.g., Oceanospirillales, Rhodospirillales, and Rhodobacterales). These new bacterial groups included putative hydrocarbon degraders, similar to those identified near the oil plume offshore. Shifts in the microbial community composition strongly correlated to more poorly sorted sediment and grain size distributional changes. Natural oceanographic processes could not account for the disrupted sediment, especially from the backshore well above the maximum high-tide levels recorded at these sites. Sand washing and tilling occurred on both open beaches from August through at least December 2010, which were mechanisms that could replace fecal indicator groups with open-ocean groups. Consequently, remediation efforts meant to return beaches to pre-spill compositions caused a regime shift that may have added potential ecosystem function, like hydrocarbon degradation, to the sediment. Future research will need to assess the persistence and impact of the newly formed microbial communities to the overall sandy beach ecosystems.

The alligator gut microbiome and implications for archosaur symbioses.

Among vertebrate gastrointestinal microbiome studies, complete representation of taxa is limited, particularly among reptiles. Here, we provide evidence for previously unrecognized host-microbiome associations along the gastrointestinal tract from the American alligator, a crown archosaur with shared ancestry to extinct taxa, including dinosaurs. Microbiome compositional variations reveal that the digestive system consists of multiple, longitudinally heterogeneous microbiomes that strongly correlate to specific gastrointestinal tract organs, regardless of rearing histories or feeding status. A core alligator gut microbiome comprised of Fusobacteria, but depleted in Bacteroidetes and Proteobacteria common to mammalians, is compositionally unique from other vertebrate gut microbiomes, including other reptiles, fish, and herbivorous and carnivorous mammals. As such, modern alligator gut microbiomes advance our understanding of archosaur gut microbiome evolution, particularly if conserved host ecology has retained archosaur-specific symbioses over geologic time.

A unique midgut-associated bacterial community hosted by the cave beetle Cansiliella servadeii (Coleoptera: Leptodirini) reveals parallel phylogenetic divergences from universal gut-specific ancestors.

BACKGROUND: Cansiliella servadeii (Coleoptera) is an endemic troglobite living in deep carbonate caves in North-Eastern Italy. The beetle constantly moves and browses in its preferred habitat (consisting in flowing water and moonmilk, a soft speleothem colonized by microorganisms) self-preens to convey material from elytra, legs, and antennae towards the mouth. We investigated its inner and outer microbiota using microscopy and DNA-based approaches. RESULTS: Abundant microbial cell masses were observed on the external appendages. Cansiliella's midgut is fully colonized by live microbes and culture-independent analyses yielded nearly 30 different 16S phylotypes that have no overlap with the community composition of the moonmilk. Many of the lineages, dominated by Gram positive groups, share very low similarity to database sequences. However for most cases, notwithstanding their very limited relatedness with existing records, phylotypes could be assigned to bacterial clades that had been retrieved from insect or other animals' digestive traits. CONCLUSIONS: Results suggest a history of remote separation from a common ancestor that harboured a set of gut-specific bacteria whose functions are supposedly critical for host physiology. The phylogenetic and coevolutionary implications of the parallel occurrences of these prokaryotic guilds appear to apply throughout a broad spectrum of animal diversity. Their persistence and conservation underlies a possibly critical role of precise bacterial assemblages in animal-bacteria interactions.

Arsenite oxidase gene diversity among Chloroflexi and Proteobacteria from El Tatio Geyser Field, Chile.

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 (aioA) 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 aioA-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 Deinococci, Thermus, Aquificales, or Chlorobi aioA-like genes were retrieved. The functional importance of amino acid sites was evaluated from evolutionary trace analyses of all retrieved aioA 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.

Evidence for niche partitioning revealed by the distribution of sulfur oxidation genes collected from areas of a terrestrial sulfidic spring with differing geochemical conditions.

The diversity and phylogenetic significance of bacterial genes in the environment has been well studied, but comparatively little attention has been devoted to understanding the functional significance of different variations of the same metabolic gene that occur in the same environment. We analyzed the geographic distribution of 16S rRNA pyrosequences and soxB genes along a geochemical gradient in a terrestrial sulfidic spring to identify how different taxonomic variations of the soxB gene were naturally distributed within the spring outflow channel and to identify possible evidence for altered SoxB enzyme function in nature. Distinct compositional differences between bacteria that utilize their SoxB enzyme in the Paracoccus sulfide oxidation pathway (e.g., Bradyrhizobium, Paracoccus, and Rhodovulum) and bacteria that utilize their SoxB enzyme in the branched pathway (e.g., Chlorobium, Thiothrix, Thiobacillus, Halothiobacillus, and Thiomonas) were identified. Different variations of the soxB genes were present at different locations within the spring outflow channel in a manner that significantly corresponded to geochemical conditions. The distribution of the different soxB gene sequence variations suggests that the enzymes encoded by these genes are functionally different and could be optimized to specific geochemical conditions that define niche space for bacteria capable of oxidizing reduced sulfur compounds.

Linking phylogenetic and functional diversity to nutrient spiraling in microbial mats from Lower Kane Cave (USA).

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.

Diversity of uncultured Epsilonproteobacteria from terrestrial sulfidic caves and springs.

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.

Speciation of sulfur from filamentous microbial mats from sulfidic cave springs using X-ray absorption near-edge spectroscopy.

Most transformations within the sulfur cycle are controlled by the biosphere, and deciphering the abiotic and biotic nature and turnover of sulfur is critical to understand the geochemical and ecological changes that have occurred throughout the Earth's history. Here, synchrotron radiation-based sulfur K-edge X-ray absorption near-edge structure (XANES) spectroscopy is used to examine sulfur speciation in natural microbial mats from two aphotic (cave) settings. Habitat geochemistry, microbial community compositions, and sulfur isotope systematics were also evaluated. Microorganisms associated with sulfur metabolism dominated the mats, including members of the Epsilonproteobacteria and Gammaproteobacteria. These groups have not been examined previously by sulfur K-edge XANES. All of the mats consisted of elemental sulfur, with greater contributions of cyclo-octasulfur (S8) compared with polymeric sulfur (Smicro). While this could be a biological fingerprint for some bacteria, the signature may also indicate preferential oxidation of Smicro and S8 accumulation. Higher sulfate content correlated to less S8 in the presence of Epsilonproteobacteria. Sulfur isotope compositions confirmed that sulfur content and sulfur speciation may not correlate to microbial metabolic processes in natural samples, thereby complicating the interpretation of modern and ancient sulfur records.

The versatile epsilon-proteobacteria: key players in sulphidic habitats.

The epsilon-proteobacteria have recently been recognized as globally ubiquitous in modern marine and terrestrial ecosystems, and have had a significant role in biogeochemical and geological processes throughout Earth's history. To place this newly expanded group, which consists mainly of uncultured representatives, in an evolutionary context, we present an overview of the taxonomic classification for the class, review ecological and metabolic data in key sulphidic habitats and consider the ecological and geological potential of the epsilon-proteobacteria in modern and ancient systems. These integrated perspectives provide a framework for future culture- and genomic-based studies.

Bacterial diversity and ecosystem function of filamentous microbial mats from aphotic (cave) sulfidic springs dominated by chemolithoautotrophic "Epsilonproteobacteria".

Filamentous microbial mats from three aphotic sulfidic springs in Lower Kane Cave, Wyoming, were assessed with regard to bacterial diversity, community structure, and ecosystem function using a 16S rDNA-based phylogenetic approach combined with elemental content and stable carbon isotope ratio analyses. The most prevalent mat morphotype consisted of white filament bundles, with low C:N ratios (3.5-5.4) and high sulfur content (16.1-51.2%). White filament bundles and two other mat morphotypes had organic carbon isotope values (mean delta13C=-34.7 per thousand, 1sigma=3.6) consistent with chemolithoautotrophic carbon fixation from a dissolved inorganic carbon reservoir (cave water, mean delta13C=-7.4 per thousand for two springs, n=8). Bacterial diversity was low overall in the clone libraries, and the most abundant taxonomic group was affiliated with the "Epsilonproteobacteria" (68%), with other bacterial sequences affiliated with Gammaproteobacteria (12.2%), Betaproteobacteria (11.7%), Deltaproteobacteria (0.8%), and the Acidobacterium (5.6%) and Bacteriodetes/Chlorobi (1.7%) divisions. Six distinct epsilonproteobacterial taxonomic groups were identified from the microbial mats. Epsilonproteobacterial and bacterial group abundances and community structure shifted from the spring orifices downstream, corresponding to changes in dissolved sulfide and oxygen concentrations and metabolic requirements of certain bacterial groups. Most of the clone sequences for epsilonproteobacterial groups were retrieved from areas with high sulfide and low oxygen concentrations, whereas Thiothrix spp. and Thiobacillus spp. had higher retrieved clone abundances where conditions of low sulfide and high oxygen concentrations were measured. Genetic and metabolic diversity among the "Epsilonproteobacteria" maximizes overall cave ecosystem function, and these organisms play a significant role in providing chemolithoautotrophic energy to the otherwise nutrient-poor cave habitat. Our results demonstrate that sulfur cycling supports subsurface ecosystems through chemolithoautotrophy and expand the evolutionary and ecological views of "Epsilonproteobacteria" in terrestrial habitats.

Filamentous "Epsilonproteobacteria" dominate microbial mats from sulfidic cave springs.

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.