Mycobacterium parascrofulaceum in Acidic Hot Springs in Yellowstone National Park

Mycobacterium parascrofulaceum was found in Norris Geyser Basin, Yellowstone National Park, in a system composed of two acidic (pH 3.0) springs with temperatures between 56°C at the source and 40°C at the confluence of both springs. Growth and survival assays at 56°C for 60 days were performed, confirming the origin of the strain.     

Phototrophic Phylotypes Dominate Mesothermal Microbial Mats Associated with Hot Springs in Yellowstone National Park

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.     

Archaeal and Bacterial Glycerol Dialkyl Glycerol Tetraether Lipids in Hot Springs of Yellowstone National Park

Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids originally thought to be produced mainly by (hyper)thermophilic archaea. Environmental screening of low-temperature environments showed, however, the abundant presence of structurally diverse GDGTs from both bacterial and archaeal sources. In this study, we examined the occurrences and distribution of GDGTs in hot spring environments in Yellowstone National Park with high temperatures (47 to 83°C) and mostly neutral to alkaline pHs. GDGTs with 0 to 4 cyclopentane moieties were dominant in all samples and are likely derived from both (hyper)thermophilic Crenarchaeota and Euryarchaeota. GDGTs with 4 to 8 cyclopentane moieties, likely derived from the crenarchaeotal order Sulfolobales and the euryarchaeotal order Thermoplasmatales, are usually present in much lower abundance, consistent with the relatively high pH values of the hot springs. The relative abundances of cyclopentane-containing GDGTs did not correlate with in situ temperature and pH, suggesting that other environmental and possibly genetic factors play a role as well. Crenarchaeol, a biomarker thought to be specific for nonthermophilic group I Crenarchaeota, was also found in most hot springs, though in relatively low concentrations, i.e., <5% of total GDGTs. Its abundance did not correlate with temperature, as has been reported previously. Instead, the cooccurrence of relatively abundant nonisoprenoid GDGTs thought to be derived from soil bacteria suggests a predominantly allochthonous source for crenarchaeol in these hot spring environments. Finally, the distribution of bacterial branched GDGTs suggests that they may be derived from the geothermally heated soils surrounding the hot springs.     

Isolation and Distribution of a Novel Iron-Oxidizing Crenarchaeon from Acidic Geothermal Springs in Yellowstone National Park

Novel thermophilic crenarchaea have been observed in Fe(III) oxide microbial mats of Yellowstone National Park (YNP); however, no definitive work has identified specific microorganisms responsible for the oxidation of Fe(II). The objectives of the current study were to isolate and characterize an Fe(II)-oxidizing member of the Sulfolobales observed in previous 16S rRNA gene surveys and to determine the abundance and distribution of close relatives of this organism in acidic geothermal springs containing high concentrations of dissolved Fe(II). Here we report the isolation and characterization of the novel, Fe(II)-oxidizing, thermophilic, acidophilic organism Metallosphaera sp. strain MK1 obtained from a well-characterized acid-sulfate-chloride geothermal spring in Norris Geyser Basin, YNP. Full-length 16S rRNA gene sequence analysis revealed that strain MK1 exhibits only 94.9 to 96.1% sequence similarity to other known Metallosphaera spp. and less than 89.1% similarity to known Sulfolobus spp. Strain MK1 is a facultative chemolithoautotroph with an optimum pH range of 2.0 to 3.0 and an optimum temperature range of 65 to 75°C. Strain MK1 grows optimally on pyrite or Fe(II) sorbed onto ferrihydrite, exhibiting doubling times between 10 and 11 h under aerobic conditions (65°C). The distribution and relative abundance of MK1-like 16S rRNA gene sequences in 14 acidic geothermal springs containing Fe(III) oxide microbial mats were evaluated. Highly related MK1-like 16S rRNA gene sequences (>99% sequence similarity) were consistently observed in Fe(III) oxide mats at temperatures ranging from 55 to 80°C. Quantitative PCR using Metallosphaera-specific primers confirmed that organisms highly similar to strain MK1 comprised up to 40% of the total archaeal community at selected sites. The broad distribution of highly related MK1-like 16S rRNA gene sequences in acidic Fe(III) oxide microbial mats is consistent with the observed characteristics and growth optima of Metallosphaera-like strain MK1 and emphasizes the importance of this newly described taxon in Fe(II) chemolithotrophy in acidic high-temperature environments of YNP.     

Microbial Composition of Near-Boiling Silica-Depositing Thermal Springs throughout Yellowstone National Park

The extent of hyperthermophilic microbial diversity associated with siliceous sinter (geyserite) was characterized in seven near-boiling silica-depositing springs throughout Yellowstone National Park using environmental PCR amplification of small-subunit rRNA genes (SSU rDNA), large-subunit rDNA, and the internal transcribed spacer (ITS). We found that Thermocrinis ruber, a member of the order Aquificales, is ubiquitous, an indication that primary production in these springs is driven by hydrogen oxidation. Several other lineages with no known close relatives were identified that branch among the hyperthermophilic bacteria. Although they all branch deep in the bacterial tree, the precise phylogenetic placement of many of these lineages is unresolved at this time. While some springs contained a fair amount of phylogenetic diversity, others did not. Within the same spring, communities in the subaqueous environment were not appreciably different than those in the splash zone at the edge of the pool, although a greater number of phylotypes was found along the pool's edge. Also, microbial community composition appeared to have little correlation with the type of sinter morphology. The number of cell morphotypes identified by fluorescence in situ hybridization and scanning electron microscopy was greater than the number of phylotypes in SSU clone libraries. Despite little variation in Thermocrinis ruber SSU sequences, abundant variation was found in the hypervariable ITS region. The distribution of ITS sequence types appeared to be correlated with distinct morphotypes of Thermocrinis ruber in different pools. Therefore, species- or subspecies-level divergences are present but not detectable in highly conserved SSU sequences.     

Isolation, Characterization, and Ecology of Sulfur-Respiring Crenarchaea Inhabiting Acid-Sulfate-Chloride-Containing Geothermal Springs in Yellowstone National Park

Elemental sulfur (S⁰) is associated with many geochemically diverse hot springs, yet little is known about the phylogeny, physiology, and ecology of the organisms involved in its cycling. Here we report the isolation, characterization, and ecology of two novel, S⁰-reducing Crenarchaea from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72°C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81°C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S⁰, and anaerobic conditions for respiration-dependent growth. Glycerol dialkyl glycerol tetraethers (GDGTs) containing four to six cyclopentyl rings were present in the lipid fraction of isolates 18U65 and 18D70. Physiological characterization suggests that the isolates are adapted to the physicochemical conditions of Dragon Spring and can utilize the natural organic matter in the spring as a carbon and energy source. Quantitative PCR analysis of 16S rRNA genes associated with the S⁰ flocs recovered from several acid geothermal springs using isolate-specific primers indicates that these two populations together represent 17 to 37% of the floc-associated DNA. The physiological characteristics of isolates 18U65 and 18D70 are consistent with their potential widespread distribution and putative role in the cycling of sulfur in acid geothermal springs throughout the Yellowstone National Park geothermal complex. Based on phenotypic and genetic characterization, the designations Caldisphaera draconis sp. nov. and Acidilobus sulfurireducens sp. nov. are proposed for isolates 18U65 and 18D70, respectively.     

Bar-Coded Pyrosequencing Reveals Shared Bacterial Community Properties along the Temperature Gradients of Two Alkaline Hot Springs in Yellowstone National Park

An understanding of how communities are organized is a fundamental goal of ecology but one which has historically been elusive for microbial systems. We used a bar-coded pyrosequencing approach targeting the V3 region of the bacterial small-subunit rRNA gene to address the factors that structure communities along the thermal gradients of two alkaline hot springs in the Lower Geyser Basin of Yellowstone National Park. The filtered data set included a total of nearly 34,000 sequences from 39 environmental samples. Each was assigned to one of 391 operational taxonomic units (OTUs) identified by their unique V3 sequence signatures. Although the two hot springs differed in their OTU compositions, community resemblance and diversity changed with strikingly similar dynamics along the two outflow channels. Two lines of evidence suggest that these community properties are controlled primarily by environmental temperature. First, community resemblance decayed exponentially with increasing differences in temperature between samples but was only weakly correlated with physical distance. Second, diversity decreased with increasing temperature at the same rate along both gradients but was uncorrelated with other measured environmental variables. This study also provides novel insights into the nature of the ecological interactions among important taxa in these communities. A strong negative association was observed between cyanobacteria and the Chloroflexi, which together accounted for ∼70% of the sequences sampled. This pattern contradicts the longstanding hypothesis that coadapted lineages of these bacteria maintain tightly cooccurring distributions along these gradients as a result of a producer-consumer relationship. We propose that they instead compete for some limiting resource(s).Elucidating how biodiversity is distributed and the mechanisms underlying those patterns is a central goal of ecology. Although microorganisms make critical contributions to ecosystem function through their participation in biogeochemical cycles, we still have only a limited understanding of the factors that control the spatial structure and diversity of microbial communities (37). For example, although it is clear that microbial community composition is influenced by environmental variation (11, 17, 18, 28), the question of how diversity changes along environmental gradients remains generally unresolved. Of particular interest is the relationship between microbial diversity and temperature, as this environmental variable is strongly correlated with diversity over a broad range of spatial scales for many plant and animal taxa in terrestrial, freshwater, and marine ecosystems (1, 12, 36, 39, 43). In addition, because spatially resolved abundance data for individual microbial taxa are scarce, we have only limited information regarding the patterns of association among microorganisms. Consequently, microbial ecology has developed with little clarity regarding the potential roles of either negative or positive biotic interactions for structuring communities or whether microbial community organization along environmental gradients conforms to either individualistic (e.g., see references 13 and 20) or organismal (e.g., see reference 9) community ecology paradigms developed for macroscopic organisms.Hot spring microbial ecosystems present an excellent opportunity to investigate fundamental questions regarding community organization. Steep temperature gradients enable the explicit investigation of the importance of environmental variation for structuring diversity while controlling for other factors that typically vary across sampling locations on larger geographic scales, such as solar energy availability and geological history. Decades of biological research on the alkaline-silica hot springs of Yellowstone National Park (reviewed in reference 49) further inform predictions regarding the nature of the ecological interactions among abundant community members.These systems are particularly notable for the presence of ecologically diverse groups of cyanobacteria and photosynthetic green nonsulfur bacteria (i.e., the Chloroflexi). The former group includes lineages of Synechococcus, the most thermotolerant of which delimits the thermal maximum for photosynthesis, whereas the latter includes divergent “green” (the genus Chloroflexus and relatives) and “red” (the genus Roseiflexus) clades. The conventional view is that interactions among cyanobacteria and the Chloroflexi are generally positive. Specifically, it has been proposed that coadapted lineages of Synechococcus and Chloroflexi maintain tightly cooccurring distributions due to a producer-consumer relationship in which the Chloroflexi grow as photoheterotrophs on low-molecular-weight organic compounds excreted by the cyanobacteria (48, 49). In turn, the filamentous Chloroflexi were previously suggested to provide a matrix within which Synechococcus cells become stably embedded (5). According to this model, we would expect to observe coincident peaks in abundance between coadapted lineages of cyanobacteria and Chloroflexi along alkaline hot spring gradients as well as a general positive correlation between the abundances of both groups. However, other evidence raises the possibility that these groups may not share a strict cross-feeding relationship. The genus Chloroflexus is metabolically flexible in laboratory culture, and certain strains have been grown as photoautotrophs with hydrogen sulfide or hydrogen as an electron donor (16, 26, 30). Although Roseiflexus is yet to be grown autotrophically in the laboratory, comparative genomics of laboratory strains and metagenomic data from microbial mat communities have also revealed the presence of Roseiflexus genes involved in the autotrophic hydroxypropionate pathway (24). In addition, stable carbon isotope data (46, 47) suggest that certain members of the Chloroflexi may have the capacity to grow autotrophically in situ. The issue of the nature of the ecological interactions between cyanobacteria and the Chloroflexi therefore requires clarification.In the present study, we investigated the patterns of distribution of bacteria along the temperature gradients of two alkaline-silica hot springs in Yellowstone National Park to determine how community properties changed in response to temperature and whether the realized distributions of cyanobacteria and the Chloroflexi meet the prediction of the coadaptation hypothesis. To do so, we used bar-coded mass parallel pyrosequencing of the V3 variable region of the bacterial small-subunit (SSU) rRNA gene to simultaneously interrogate the sequence diversity of environmental samples from multiple locations along both hot springs. This strategy is distinct from recent applications of next-generation DNA sequencing technology for investigating microbial diversity, as previous efforts focused principally on the deep sampling of one or a few discrete habitats for the purpose of quantifying the magnitude of diversity (e.g., see references 21, 38, and 41). We report that community properties changed with similar dynamics in response to temperature despite differences between hot springs in water chemistries and taxon composition, and we reject the coadaptation hypothesis for these communities based on a strong negative association in abundances of cyanobacteria and Chloroflexi.     

The Role of Extracellular Polymeric Substances in the Silicification of Calothrix: Evidence from Microbial Mat Communities in Hot Springs at Yellowstone National Park,USA

We provide nanoscale evidence of the role of sheath exopolymers in the silicification of the sheathed cyanobacteria Calothrix. Electron microscope observations of silicified Calothrix cells revealed that silica accretes directly onto EPS sheath fibrils to produce an open web of silica particles that could remain permeable to nutrients and waste products. We also found that silicified Calothrix cells from different microhabitats contained morphologically distinct silica particles. Differences in silicification texture suggest that environmental variables may influence silicification at the nanoscale. We develop a framework based on aggregation kinetics to address silicification processes in Calothrix and other sheathed cyanobacteria.     

Hot spring siliceous stromatolites from Yellowstone National Park: assessing growth rate and laminae formation

Stromatolites are commonly interpreted as evidence of ancient microbial life, yet stromatolite morphogenesis is poorly understood. We apply radiometric tracer and dating techniques, molecular analyses and growth experiments to investigate siliceous stromatolite morphogenesis in Obsidian Pool Prime (OPP), a hot spring in Yellowstone National Park. We examine rates of stromatolite growth and the environmental and/or biologic conditions that affect lamination formation and preservation, both difficult features to constrain in ancient examples. The "main body" of the stromatolite is composed of finely laminated, porous, light-dark couplets of erect (surface normal) and reclining (surface parallel) silicified filamentous bacteria, interrupted by a less-distinct, well-cemented "drape" lamination. Results from dating studies indicate a growth rate of 1-5 cm year(-1) ; however, growth is punctuated. (14)C as a tracer demonstrates that stromatolite cyanobacterial communities fix CO(2) derived from two sources, vent water (radiocarbon dead) and the atmosphere (modern (14)C). The drape facies contained a greater proportion of atmospheric CO(2) and more robust silica cementation (vs. the main body facies), which we interpret as formation when spring level was lower. Systematic changes in lamination style are likely related to environmental forcing and larger scale features (tectonic, climatic). Although the OPP stromatolites are composed of silica and most ancient forms are carbonate, their fine lamination texture requires early lithification. Without early lithification, whether silica or carbonate, it is unlikely that a finely laminated structure representing an ancient microbial mat would be preserved. In OPP, lithification on the nearly diurnal time scale is likely related to temperature control on silica solubility.     

A geothermal‐linked biological oasis in Yellowstone Lake,Yellowstone National Park,Wyoming

Hundreds of active and dormant geothermal vents have been located on the floor of Yellowstone Lake, although characterization of the associated biology (macro or micro) has been extremely limited. Herein, we describe an aquatic moss (Fontinalis) colony closely associated with vent emissions that considerably exceeded known temperature maxima for this plant. Vent waters were supersaturated with CO₂, likely accommodating a CO₂ compensation point that would be expected to be quite elevated under these conditions. The moss was colonized by metazoa, including the crustaceans Hyalella and Gammarus, a segmented worm in the Lumbriculidae family, and a flatworm specimen tentatively identified as Polycelis. The presence of these invertebrates suggest a highly localized food chain that derives from the presence of geothermal inputs and thus is analogous to the deep marine vents that support significant biodiversity.     

PCR Detection and Analysis of the Free-Living Amoeba Naegleria in Hot Springs in Yellowstone and Grand Teton National Parks

Free-living thermotolerant amoebae pose a significant health risk to people who soak and swim in habitats suitable for their growth, such as hot springs. In this survey of 23 different hot springs in Yellowstone and Grand Teton National Parks, we used PCR with primer sets specific for Naegleria to detect three sequence types that represent species not previously described, as well as a fourth sequence type identified as the pathogen Naegleria fowleri.     

Diversity and Functional Analysis of Bacterial Communities Associated with Natural Hydrocarbon Seeps in Acidic Soils at Rainbow Springs, Yellowstone National Park

In this paper we describe the bacterial communities associated with natural hydrocarbon seeps in nonthermal soils at Rainbow Springs, Yellowstone National Park. Soil chemical analysis revealed high sulfate concentrations and low pH values (pH 2.8 to 3.8), which are characteristic of acid-sulfate geothermal activity. The hydrocarbon composition of the seep soils consisted almost entirely of saturated, acyclic alkanes (e.g., n-alkanes with chain lengths of C₁₅ to C₃₀, as well as branched alkanes, predominately pristane and phytane). Bacterial populations present in the seep soils were phylogenetically characterized by 16S rRNA gene clone library analysis. The majority of the sequences recovered (>75%) were related to sequences of heterotrophic acidophilic bacteria, including Acidisphaera spp. and Acidiphilium spp. of the α-Proteobacteria. Clones related to the iron- and sulfur-oxidizing chemolithotroph Acidithiobacillus spp. were also recovered from one of the seep soils. Hydrocarbon-amended soil-sand mixtures were established to examine [¹⁴C]hexadecane mineralization and corresponding changes in the bacterial populations using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Approximately 50% of the [¹⁴C]hexadecane added was recovered as ¹⁴CO₂ during an 80-day incubation, and this was accompanied by detection of heterotrophic acidophile-related sequences as dominant DGGE bands. An alkane-degrading isolate was cultivated, whose 16S rRNA gene sequence was identical to the sequence of a dominant DGGE band in the soil-sand mixture, as well as the clone sequence recovered most frequently from the original soil. This and the presence of an alkB gene homolog in this isolate confirmed the alkane degradation capability of one population indigenous to acidic hydrocarbon seep soils.     

Diversity and functional analysis of bacterial communities associated with natural hydrocarbon seeps in acidic soils at Rainbow Springs, Yellowstone National Park

In this paper we describe the bacterial communities associated with natural hydrocarbon seeps in nonthermal soils at Rainbow Springs, Yellowstone National Park. Soil chemical analysis revealed high sulfate concentrations and low pH values (pH 2.8 to 3.8), which are characteristic of acid-sulfate geothermal activity. The hydrocarbon composition of the seep soils consisted almost entirely of saturated, acyclic alkanes (e.g., n-alkanes with chain lengths of C15 to C30, as well as branched alkanes, predominately pristane and phytane). Bacterial populations present in the seep soils were phylogenetically characterized by 16S rRNA gene clone library analysis. The majority of the sequences recovered (>75%) were related to sequences of heterotrophic acidophilic bacteria, including Acidisphaera spp. and Acidiphilium spp. of the alpha-Proteobacteria. Clones related to the iron- and sulfur-oxidizing chemolithotroph Acidithiobacillus spp. were also recovered from one of the seep soils. Hydrocarbon-amended soil-sand mixtures were established to examine [14C]hexadecane mineralization and corresponding changes in the bacterial populations using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Approximately 50% of the [14C]hexadecane added was recovered as 14CO2 during an 80-day incubation, and this was accompanied by detection of heterotrophic acidophile-related sequences as dominant DGGE bands. An alkane-degrading isolate was cultivated, whose 16S rRNA gene sequence was identical to the sequence of a dominant DGGE band in the soil-sand mixture, as well as the clone sequence recovered most frequently from the original soil. This and the presence of an alkB gene homolog in this isolate confirmed the alkane degradation capability of one population indigenous to acidic hydrocarbon seep soils.     

Fungi from Geothermal Soils in Yellowstone National Park

Geothermal soils near Amphitheater Springs in Yellowstone National Park were characterized by high temperatures (up to 70°C), high heavy metal content, low pH values (down to pH 2.7), sparse vegetation, and limited organic carbon. From these soils we cultured 16 fungal species. Two of these species were thermophilic, and six were thermotolerant. We cultured only three of these species from nearby cool (0 to 22°C) soils. Transect studies revealed that higher numbers of CFUs occurred in and below the root zone of the perennial plant Dichanthelium lanuginosum (hot springs panic grass). The dynamics of fungal CFUs in geothermal soil and nearby nongeothermal soil were investigated for 12 months by examining soil cores and in situ mesocosms. For all of the fungal species studied, the temperature of the soil from which the organisms were cultured corresponded with their optimum axenic growth temperature.     

Fungi from geothermal soils in Yellowstone National Park

Geothermal soils near Amphitheater Springs in Yellowstone National Park were characterized by high temperatures (up to 70 degrees C), high heavy metal content, low pH values (down to pH 2.7), sparse vegetation, and limited organic carbon. From these soils we cultured 16 fungal species. Two of these species were thermophilic, and six were thermotolerant. We cultured only three of these species from nearby cool (0 to 22 degrees C) soils. Transect studies revealed that higher numbers of CFUs occurred in and below the root zone of the perennial plant Dichanthelium lanuginosum (hot springs panic grass). The dynamics of fungal CFUs in geothermal soil and nearby nongeothermal soil were investigated for 12 months by examining soil cores and in situ mesocosms. For all of the fungal species studied, the temperature of the soil from which the organisms were cultured corresponded with their optimum axenic growth temperature.     

Division-Specific Differences in Bacterial Community Assembly of Two Alkaline Hot Spring Ecosystems from Yellowstone National Park

A fundamental issue in ecology is whether communities are random assemblages or, alternatively, whether there are rules that determine which combinations of taxa can co-occur. For microbial systems, in particular, the question of whether taxonomic groups exhibit differences in community organization remains unresolved but is critical for our understanding of community structure and function. Here, we used presence–absence matrices derived from bar-coded pyrosequencing data to evaluate the assembly patterns of eight bacterial divisions distributed along two Yellowstone National Park hot spring outflow channels. Four divisions (Cyanobacteria, Chloroflexi, Acidobacteria, and Cytophaga–Flavobacterium–Bacteroides) exhibited less co-occurrence than expected by chance, with phototrophic taxa showing the strongest evidence for nonrandom community structure. We propose that both differences in environmental tolerance and competitive interactions within divisions contribute to these nonrandom assembly patterns. The higher degree of nonrandom structure observed for phototrophic taxa compared with the other divisions may be due in part to greater overlap in resource usage, as has been previously proposed for plant communities.     

Elemental analysis of mineral lick soils from the Serengeti National Park, the Konza Prairie and Yellowstone National Park

We analyzed mineral lick soils and non-mineral lick soils from the Serengeti National Park, the Konza Praine, and Yellowstone National Park The concentrations of 14 different elements considered important for ungulate nutrition were analyzed to determine the relative importance of Na vs other elements in attracting animals to lick areas Only Na was consistently higher in the mineral lick soils of the three ecosystems Mean Na concentrations were c 20x greater in Serengeti licks, 10x greater in Konza licks and 3x greater in Yellowstone licks compared to the respective non-lick soils Despite the numerous elements analyzed for this survey, our results suggest that Na is the primary element initiates mineral lick use in these widely separated ecosystems The widespread distribution of Na-deficient forage likely explains the importance of Na in most mineral licks