Great Salt Plains Research Articles

Bacterial Growth in Brines Formed by the Deliquescence of Salts Relevant to Cold Arid Worlds.

Hygroscopic salts at Mars' near-surface (MgSO4, (per)chlorates, NaCl) may form brines by absorbing moisture from the atmosphere at certain times through the process of deliquescence. We have previously shown strong bacterial growth in saturated MgSO4 (∼67% w/v as epsomite) at room temperature, and growth was observed at the MgSO4 eutectic point (43% w/v at -4°C). Here, we have investigated the growth of salinotolerant microbes (Halomonas, Marinococcus, Planococcus) from Hot Lake, Washington; Basque Lake, British Columbia; and Great Salt Plains, Oklahoma under deliquescing conditions. Bacterial cultures were grown to mid-log phase in SP medium supplemented with 50% MgSO4 (as epsomite), 20% NaClO3, or 10% NaCl (w/v), and small aliquots in cups were dried by vacuum desiccation. When the dried culture was rehydrated by the manual addition of water, the culture resumed growth in the reconstituted brine. When desiccated cultures were maintained in a sealed container with a brine reservoir of the matching growth medium controlling the humidity of the headspace, the desiccated microbial culture evaporites formed brine by deliquescence using humidity alone. Bacterial cultures resumed growth in all three salts once rehydrated by deliquescence. Cultures of Halomonas sp. str. HL12 showed robust survival and growth when subjected to several cycles of desiccation and deliquescent or manual rehydration. Our laboratory demonstrations of microbial growth in deliquescent brines are relevant to the surface and near-subsurface of cold arid worlds like Mars. When conditions become wetter, hygroscopic evaporite minerals can deliquesce to produce the earliest habitable brines. Survival after desiccation and growth in deliquescent brines increases the likelihood that microbes from Earth, carried on spacecraft, pose a contamination risk to Mars.

Molecular Characterization of Plasmids Harbored by Actinomycetes Isolated From the Great Salt Plains of Oklahoma Using PFGE and Next Generation Whole Genome Sequencing.

One of the unique features of actinomycetes, especially the genus Streptomyces, is the presence of linear plasmids. These range in size from 12 to 600 kb, and are often termed mega-plasmids. While many of the genes involved in secondary metabolite production reside in clusters on the chromosome, several studies have identified biosynthetic clusters on large linear plasmids that produce important secondary metabolites, including antibiotics. In this study, Pulse Field Gel Electrophoresis (PFGE) was used to screen 176 actinomycete isolates for the presence of plasmids; these bacterial strains were previously isolated from the Great Salt Plains of Oklahoma. Seventy-eight of the 176 actinomycete isolates (44%) contained plasmids. Several strains contained more than one plasmid, accounting for a total of 109 plasmids. Ten isolates showed extrachromosomal DNA larger than 200 kb, thus falling into the category of mega-plasmids. A subset of plasmids from 55 isolates was treated with S1 nuclease to determine topology; all plasmids examined appeared to be linear and ranged from ~55 to 400 kb. Eleven isolates were chosen for Whole Genome Next Generation Sequencing. From the 11 sequenced isolates, seven plasmids were partially assembled. While the majority of the genes identified on the plasmids coded for hypothetical proteins, others coded for general functions, stress response, and antibiotic and heavy metal resistance. Draft genome sequences of two mega-plasmid-bearing Streptomyces sp. strains, BF-3 and 4F, revealed the presence of genes involved in antibiotic production, antibiotic, and heavy metal resistance, osmoregulation, and stress response, which likely facilitate their survival in this extreme halophilic environment. To our knowledge, this is the first study to explore plasmids harbored by actinomycetes isolated from the Great Salt Plains of Oklahoma.

Draft Genome Sequences of Megaplasmid-Bearing Streptomyces sp. Strains BF-3 and 4F, Isolated from the Great Salt Plains of Oklahoma.

ABSTRACTDraft genome sequences of megaplasmid-bearing Streptomyces sp. strains BF-3 and 4F, isolated from the Great Salt Plains of Oklahoma, showed genome sizes of 7,950,134 and 7,550,992 bp, respectively. Both genomes revealed the presence of genes involved in osmoregulation and stress response, potentially helping their survival in such an extreme environment.

The Whole-Genome Sequence of Bacillus velezensis Strain SB1216 Isolated from the Great Salt Plains of Oklahoma Reveals the Presence of a Novel Extracellular RNase with Antitumor Activity

ABSTRACTThe whole-genome sequence of Bacillus velezensis strain SB1216, isolated from the Great Salt Plains of Oklahoma, showed the presence of a 3,814,720-bp circular chromosome and no plasmids. The presence of a novel 870-bp extracellular RNase gene is predicted to be responsible for this strain’s antitumor activity.

Acceptability ofDiorhabda carinulata, an Introduced Biological Control Agent ofTamarix, as Prey for Selected Resident Native Arthropod Predators

Although biological control agents are imported without the constraints exerted by their native natural enemies, many native, generalist arthropod predators might use the new food source after release in the field. Predation by native arthropods is a potential obstacle to establishment of Diorhabda spp. as a biological control agent against saltcedar, Tamarix spp. Arboreal predaceous insects were surveyed at the Great Salt Plains National Wildlife Refuge to identify possible predators that could affect establishment of the northern tamarisk beetle, Diorhabda carinulata (Desbrochers), in Oklahoma. Results of the survey showed that 57% of the arthropods were represented by five insects, Zelus tetracanthus (Stal) (Hemiptera: Reduviidae) (21%); several species of Chrysoperla (Neuroptera: Chrysopidae) (11%); the insidious flower bug, Orius insidiosus (Say) (Hemiptera: Anthocoridae) (10%); Collops quadrimaculatus (F.) Coleoptera: Melydridae) (8%); and the sevenspotted lady beetle, Coccinella septempunctata L. (Coleoptera: Coccinellidae) (6%), in saltcedar stands. No-choice feeding studies demonstrated that eggs and larvae of D. carinulata were accepted prey for larvae of Chrysoperla rufilabris (Burmeister) and sevenspotted lady beetle adults but not larvae. Northern tamarisk beetle larvae were accepted prey for Z. tetracanthus.

Characterization of Green Microalgae and Cyanobacteria Isolated from the Great Salt Plains

<abstract> The objective of this study was to characterize the growth pattern, lipid content, fatty acid profile, and biomass characteristics of seven algae strains isolated from the same environment and grown in standard media under similar conditions. Proximate composition of the algae biomass was determined using a thermogravimetric analysis method. The growth profile of a selected strain (SP22) in wastewater generated during hydraulic fracturing for natural gas production (frac water) was also examined. Average biomass productivities of SP20, SP46, and SP50 were similar and significantly higher than the other strains examined in this study. Maximum biomass concentration in the culture medium (1.32 g L^-1) was achieved with SP46. Green algae strains SP20 and SP22 accumulated significantly higher amounts of lipids than all the cyanobacteria investigated in this study. Saturated fatty acid contents of the algal oils varied between 32.5% and 39.9%. The highest mono- and polyunsaturated fatty acid contents were observed in SP46 and SP22, respectively. Total FAME content was highest in SP22 and second highest in SP20. Growth potential of SP22 in wastewater was also examined. This study demonstrated that SP22 can grow in frac water. Furthermore, enrichment of frac water with nutrients resulted in about 20 times more biomass productivity than that of the frac water as is, with no nutrient addition.

Thermal Degradation and Microwave-Assisted Pyrolysis of Green Algae and Cyanobacteria Isolated from the Great Salt Plains

Seven algae strains isolated from the Great Salt Plains of Oklahoma (UTEX SP20, SP22, SP38, SP46, SP47, SP48, and SP50) were examined in this study. Biomass thermal degradation behavior of each strain was evaluated by thermogravimetric analysis. Kinetic parameters were determined using an iso-conversional approach. Algal biomass was used for bio-oil production via microwave-assisted pyrolysis, and the effects of final temperature on product yields and bio-oil composition were evaluated. Thermogravimetric analyses revealed that pyrolysis of algal biomass took place in three stages, with major weight loss occurring at the second stage from around 150°C to 400°C. Biomass of SP38 had the lowest average apparent activation energy (102.8 kJ mol-1), indicating that biomass of SP38 requires the least energy for pyrolysis among the seven strains. SP46 was selected out of the seven strains for microwave-assisted pyrolysis because of its high final biomass concentration and biomass productivity. As the final temperature increased from 450°C to 750°C, the bio-oil yield increased from 4.6% to 22.5% during microwave-assisted pyrolysis of biomass generated by SP46. The major compounds in the bio-oil were acids, aliphatic hydrocarbons, aromatic hydrocarbons, phenols, and organic nitrogen compounds. Microwave-assisted pyrolysis of algal biomass did not produce polycyclic aromatic hydrocarbons at temperatures higher than 450°C.

Bacterial growth tolerance to concentrations of chlorate and perchlorate salts relevant to Mars

AbstractThe Phoenix lander at Mars polar cap found appreciable levels of (per)chlorate salts, a mixture of perchlorate and chlorate salts of Ca, Fe, Mg and Na at levels of ~0.6% in regolith. These salts are highly hygroscopic and can form saturated brines through deliquescence, likely producing aqueous solutions with very low freezing points on Mars. To support planetary protection efforts, we have measured bacterial growth tolerance to (per)chlorate salts. Existing bacterial isolates from the Great Salt Plains of Oklahoma (NaCl-rich) and Hot Lake in Washington (MgSO4-rich) were tested in high concentrations of Mg, K and Na salts of chlorate and perchlorate. Strong growth was observed with nearly all of these salinotolerant isolates at 1% (~0.1 M) (per)chlorate salts, similar to concentrations observed in bulk soils on Mars. Growth in perchlorate salts was observed at concentrations of at least 10% (~1.0 M). Greater tolerance was observed for chlorate salts, where growth was observed to 2.75 M (&gt;25%). Tolerance to K salts was greatest, followed by Mg salts and then Na salts. Tolerances varied among isolates, even among those within the same phylogenetic clade. Tolerant bacteria included genera that also are found in spacecraft assembly facilities. Substantial microbial tolerance to (per)chlorate salts is a concern for planetary protection since tolerant microbes contaminating spacecraft would have a greater chance for survival and proliferation, despite the harsh chemical conditions found near the surface of Mars.

Relationships between sucretolerance and salinotolerance in bacteria from hypersaline environments and their implications for the exploration of Mars and the icy worlds

AbstractThe most extremely osmotolerant microbial isolates are fungi from high-sugar environments that tolerate the lowest water activity (0.61) for growth yet reported. Studies of osmotolerant bacteria have focused on halotolerance rather than sucretolerance (ability to grow in high sugar concentrations). A collection of salinotolerant (≥10% NaCl or ≥50% MgSO4) bacterial isolates from the Great Salt Plains of Oklahoma and Hot Lake in Washington were screened for sucretolerance in medium supplemented with ≥50% fructose, glucose or sucrose. Tolerances significantly differed between solutes, even though water activities for saline media (0.92 and 0.85 for 10 and 20% NaCl Salt Plains media, respectively) were comparable or lower than water activities for high-sugar media (0.93 and 0.90 for 50 and 70% sucrose artificial nectar media, respectively). These specific solute effects were differentially expressed among individual isolates. Extrapolating the results of earlier food science studies with yeasts at high sugar concentrations to bacteria in salty environments with low water activity should be done with caution. Furthermore, the discussion of habitable Special Regions on Mars and the icy worlds should reflect an understanding of specific solute effects.

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The extreme environment of the Great Salt Plains (GSP) results in low algal biomass, such that natural selection is likely driven by survival of multiple abiotic stresses rather than rapid growth and biotic interactions. The objective of this study was to determine the effect of combined salinity stress and iron limitation on growth and photosynthesis in the green alga, Picochlorum oklahomensis isolated from the GSP habitat. Algal cells were grown in batch cultures under iron sufficiency or iron limitation at salinities of 10, 50 and 100 ppt in artificial seawater (AS 100) medium. Cells were physiologically characterized by growth rates, cell density, photosynthetic light-response curves (oxygen evolution), pigment composition and the chlorophyll fluorescence parameters Fv/Fm, ФPSII, qP and NPQ. P. oklahomensis cultured in no added Fe exhibit smaller inhibition by 100 ppt salinity relative to 10 ppt. Thus, high salinity appears to be reduced under low nutrient conditions. Fe stress resulted in qualitative differences in physiological response. The interaction between salinity and low iron is consistent with a general stress response that concurrently protects against several abiotic stress factors. Key words: Iron limitation, salinity stress, Picochlorum sp.

Isolation and Characterization of Halotolerant Soil Fungi from the Great Salt Plains of Oklahoma.

The Great Salt Plains (GSP) of Oklahoma is an inland terrestrial hypersaline environment where saturated brines leave evaporite crusts of NaCl. The current report examines the fungal community, complementing earlier reports on the bacterial and archaeal communities. Twenty-five fungal isolates from GSP soils were obtained on medium containing 10% NaCl and characterized. Based on 18S rRNA gene sequence analysis, all of the isolates fall within the Ascomycetes, with a predominance of Trichocomaceae, represented by Aspergillus, Eurotium, and Penicillium species. Representatives of Anthrinium, Cladosporium, Debaryomyces, Fusarium, and Ulocladium also were isolated. Overall the isolates were widely halotolerant, with best growth observed at lower salinities and no halophilism. The fungal genera observed were all cosmopolitan, without strong specialization. Taken together, these results support the conclusion that hypersaline environments do not have a characteristic community, in contrast to what was observed at the GSP for bacteria and archaea.

Colorimetric microbial diversity analysis and halotolerance along a soil salinity gradient at the Great Salt Plains of Oklahoma

Microbial diversity was measured along a salinity gradient at the Great Salt Plains of Oklahoma using colony color quantified as RGB components of microbial isolate streaks. Numerical taxonomy was performed using a UPGMA method to create trees of relatedness, define OTUs, and calculate diversity indices. Surface soil samples along a 6-m salinity gradient (from hypersaline soil with 7.5% salinity to oligohaline rangeland soil) at WP68 were dilution-plated on SP medium of various salinities and hundreds of random colonies were collected. The salinity tolerance of isolates along the gradient was determined. From the 1364 colonies examined, 338 OTUs were defined by colony color and their distribution statistically analyzed by soil type and the salinity of enrichment media. Most colonies were shades of cream that became distinguishable based on RGB color components. Diversity indices were high overall and it is likely that the OTUs defined by colony color are below the species level, at the strain level, where the greatest diversity lies in this environment. These results are complementary to previous molecular genetic analyses of 16S rRNA clone libraries from soils at the Great Salt Plains. Great diversity at lower taxonomic levels supports the suggestion that gene flow is not highly fragmented, a result of less specialization, as expected given the highly variable salinity observed at the salt flats with rain events.

Bacterial growth at the high concentrations of magnesium sulfate found in martian soils.

The martian surface environment exhibits extremes of salinity, temperature, desiccation, and radiation that would make it difficult for terrestrial microbes to survive. Recent evidence suggests that martian soils contain high concentrations of MgSO₄ minerals. Through warming of the soils, meltwater derived from subterranean ice-rich regolith may exist for an extended period of time and thus allow the propagation of terrestrial microbes and create significant bioburden at the near surface of Mars. The current report demonstrates that halotolerant bacteria from the Great Salt Plains (GSP) of Oklahoma are capable of growing at high concentrations of MgSO₄ in the form of 2 M solutions of epsomite. The epsotolerance of isolates in the GSP bacterial collection was determined, with 35% growing at 2 M MgSO₄. There was a complex physiological response to mixtures of MgSO₄ and NaCl coupled with other environmental stressors. Growth also was measured at 1 M concentrations of other magnesium and sulfate salts. The complex responses may be partially explained by the pattern of chaotropicity observed for high-salt solutions as measured by agar gelation temperature. Select isolates could grow at the high salt concentrations and low temperatures found on Mars. Survival during repetitive freeze-thaw or drying-rewetting cycles was used as other measures of potential success on the martian surface. Our results indicate that terrestrial microbes might survive under the high-salt, low-temperature, anaerobic conditions on Mars and present significant potential for forward contamination. Stringent planetary protection requirements are needed for future life-detection missions to Mars.

Population Size of Snowy Plovers Breeding in North America

Snowy Plovers (Charadrius nivosus) may be one of the rarest shorebirds in North America yet a com- prehensive assessment of their abundance and distribution has not been completed. During 2007 and 2008, 557 discrete wetlands were surveyed and nine additional large wetland complexes sampled in Mexico and the USA. From these surveys, a population of 23,555 (95% CI = 17,299 - 29,859) breeding Snowy Plovers was estimated. Com- bining the estimate with information from areas not surveyed, the total North American population was assessed at 25,869 (95% CI = 18,917 - 32,173). Approximately 42% of all breeding Snowy Plovers in North America resided at two sites (Great Salt Lake, Utah, and Salt Plains National Wildlife Refuge, Oklahoma), and 33% of all these were on wetlands in the Great Basin (including Great Salt Lake). Also, coastal habitats in central and southern Texas supported large numbers of breeding plovers. New breeding sites were discovered in interior deserts and highlands and along the Pacific coast of Mexico; approximately 9% of the North American breeding population occurred in Mexico. Because of uncertainties about effects of climate change and current stresses to breeding habitats, the spe-

Phylogenetic Diversities and Community Structure of Members of the Extremely Halophilic Archaea (Order Halobacteriales) in Multiple Saline Sediment Habitats

We investigated the phylogenetic diversity and community structure of members of the halophilic Archaea (order Halobacteriales) in five distinct sediment habitats that experience various levels of salinity and salinity fluctuations (sediments from Great Salt Plains and Zodletone Spring in Oklahoma, mangrove tree sediments in Puerto Rico, sediment underneath salt heaps in a salt-processing plant, and sediments from the Great Salt Lake northern arm) using Halobacteriales-specific 16S rRNA gene primers. Extremely diverse Halobacteriales communities were encountered in all habitats, with 27 (Zodletone) to 37 (mangrove) different genera identified per sample, out of the currently described 38 Halobacteriales genera. With the exception of Zodletone Spring, where the prevalent geochemical conditions are extremely inhospitable to Halobacteriales survival, habitats with fluctuating salinity levels were more diverse than permanently saline habitats. Sequences affiliated with the recently described genera Halogranum, Halolamina, Haloplanus, Halosarcina, and Halorientalis, in addition to the genera Halorubrum, Haloferax, and Halobacterium, were among the most abundant and ubiquitous genera, suggesting a wide distribution of these poorly studied genera in saline sediments. The Halobacteriales sediment communities analyzed in this study were more diverse than and completely distinct from communities from typical hypersaline water bodies. Finally, sequences unaffiliated with currently described genera represented a small fraction of the total Halobacteriales communities, ranging between 2.5% (Zodletone) to 7.0% (mangrove and Great Salt Lake). However, these novel sequences were characterized by remarkably high levels of alpha and beta diversities, suggesting the presence of an enormous, yet-untapped supply of novel Halobacteriales genera within the rare biosphere of various saline ecosystems.

Culture‐independent analysis of the soil bacterial assemblage at the Great Salt Plains of Oklahoma

The Great Salt Plains (GSP) of Oklahoma is a natural inland terrestrial hypersaline environment that forms evaporite crusts of mainly NaCl. Previous work described GSP bacterial assemblages through the phylogenetic and phenetic characterization of 105 isolates from 46 phylotypes. The current report describes the same bacterial assemblages through culture-independent 16S rRNA gene clone libraries. Although from similar hypersaline mud flats, the bacterial libraries from two sites, WP3 and WP6, were quite different. The WP3 library was dominated by cyanobacteria, mainly Cyanothece and Euhalothece. The WP6 library was rich in anaerobic sulfur-cycle organisms, including abundant Desulfuromonas. This pattern likely reflects differences in abiotic factors, such as frequency of flooding and hydrologic push. While more than 100 OTUs were identified, the assemblages were not as diverse, based on Shannon indexes, as bacterial communities from oligohaline soils. Since natural inland hypersaline soils are relatively unstudied, it was not clear what kind of bacteria would be present. The bacterial assemblage is predominantly genera typically found in hypersaline systems, although some were relatives of microbes common in oligohaline and marine environments. The bacterial clones did not reflect wide functional diversity, beyond phototrophs, sulfur metabolizers, and numerous heterotrophs.

Effects of Bicarbonate Limitation and Salinity Stress on Growth and Photosynthesis in the Green Alga Picochlorum Oklahomensis

Photosynthetic organisms rarely experience optimal growth conditions in their natural habitat, and at any given time, two or more physical and chemical variables are likely to be suboptimal. The extreme environment of the Great Salt Plains (GSP) results in low algal biomass, such that natural selection is likely driven by survival of multiple abiotic stresses rather than rapid growth and biotic interactions. The objective of this study is to determine the effect of combined salinity stress and carbon limitation on growth and photosynthesis in the green alga Picochlorum okla-homensis isolated from the GSP habitat. Algal cells were grown in batch cultures under bicar-bonate sufficiency (control) or low bicarbonate at salinities of 10, 50 and100 ppt. in artificial seawater (AS 100) medium. Cells were physiologically characterized by initial growth rates, cell yields, photosynthetic light-response curves (oxygen evolution), pigment composition, and the chlorophyll fluorescence parameters Fv/Fm, �PSII, qP and NPQ. Low bicarbonate and increasing salinity from 10 to 100 ppt, as well as the bicarbonate-salinity interaction resulted in significant lower cell yields and initial growth rates. In general, high salinity resulted in significant primary effects for the majority of variables but a significant effect of bicarbonate and bicarbonate-salinity interaction were less common among the physiological variables.&lt;o:p&gt;&lt;/

HYPERSALINE SOIL SUPPORTS A DIVERSE COMMUNITY OF DUNALIELLA (CHLOROPHYCEAE)1

Numerous isolates of the green halophile Dunaliella were studied as part of a survey of microbial diversity at the Great Salt Plains (GSP) in Oklahoma, USA. The GSP is a large (65 km 2 ) salt flat with extreme temporal and spatial fluctuations in salinity and temperature. Although the flagellate halophile Dunaliella is common worldwide, nearly all cultured isolates are from saline habitats that are primarily aquatic rather than primarily terrestrial. The diverse GSP Dunaliella strains exhibit three morphotypes: a predominantly motile form, a motile form with a prominent palmelloid phase (nonmotile, mucilage rich), and a palmelloid form with a weakly motile phase. All had broad salinity optima well below typical in situ salinities at the GSP, and two of the palmelloid isolates grew as well in freshwater as in highly saline media. Molecular phylogenetic and evolutionary analyses revealed that Dunaliella from the GSP (and two similar habitats in the Great Basin, USA) are allied with D. viridis Teodor. but possess phylogenetic diversity in excess of existing global isolates from aquatic habitats. In addition, isolates from primarily terrestrial habitats exhibit statistically higher rates of nucleotide substitution than the phylogenetically homogeneous set of primarily aquatic Dunaliella taxa. We hypothesize that dynamically extreme saline soil habitats may select for different and more diverse Dunaliella lineages than more stable saline aquatic habitats. We also propose Dunaliella as a tractable microbial model for in situ testing of evolutionary and phylogeographic hypotheses.

Archaeal Diversity at the Great Salt Plains of Oklahoma Described by Cultivation and Molecular Analyses

The Great Salt Plains of Oklahoma is a natural inland terrestrial hypersaline environment that forms evaporite crusts of mainly NaCl. Previous work described the bacterial community through the characterization of 105 isolates from 46 phylotypes. The current report describes the archaeal community through both microbial isolation and culture-independent techniques. Nineteen distinct archaea were isolated, and ten were characterized phenetically. Included were isolates phylogenetically related to Haloarcula, Haloferax, Halorubrum, Haloterrigena, and Natrinema. The isolates were aerobic, non-motile, Gram-negative organisms and exhibited little capacity for fermentation. All of the isolates were halophilic, with most requiring at least 15% salinity for growth, and all grew at 30% salinity. The isolates were mainly mesothermic and could grow at alkaline pH (8.5). A 16S rRNA gene library was generated by polymerase chain reaction amplification of direct soil DNA extracts, and 200 clones were sequenced and analyzed. At 99% and 94% sequence identity, 36 and 19 operational taxonomic units (OTUs) were detected, respectively, while 53 and 22 OTUs were estimated by Chao1, respectively. Coverage was relatively high (100% and 59% at 89% and 99% sequence identity, respectively), and the Shannon Index was 3.01 at 99% sequence identity, comparable to or somewhat lower than hypersaline habitats previously studied. Only sequences from Euryarchaeota in the Halobacteriales were detected, and the strength of matches to known sequences was generally low, most near 90% sequence identity. Large clusters were observed that are related to Haloarcula and Halorubrum. More than two-thirds of the sequences were in clusters that did not have close relatives reported in public databases.

Cyanobacterial Diversity and Halotolerance in a Variable Hypersaline Environment

The Great Salt Plains (GSP) in north-central Oklahoma, USA is an expansive salt flat (approximately 65 km(2)) that is part of the federally protected Salt Plains National Wildlife Refuge. The GSP serves as an ideal environment to study the microbial diversity of a terrestrial, hypersaline system that experiences wide fluctuations in freshwater influx and diel temperature. Our study assessed cyanobacterial diversity at the GSP by focusing on the taxonomic and physiological diversity of GSP isolates, and the 16S rRNA phylogenetic diversity of isolates and environmental clones from three sites (north, central, and south). Taxonomic diversity of isolates was limited to a few genera (mostly Phormidium and Geitlerinema), but physiological diversity based on halotolerance ranges was strikingly more diverse, even between strains of the same phylotype. The phylogenetic tree revealed diversity that spanned a number of cyanobacterial lineages, although diversity at each site was dominated by only a few phylotypes. Unlike other hypersaline systems, a number of environmental clones from the GSP were members of the heterocystous lineage. Although a number of cyanobacterial isolates were close matches with prevalent environmental clones, it is not certain if these clones reflect the same halotolerance ranges of their matching isolates. This caveat is based on the notable disparities we found between strains of the same phylotype and their inherent halotolerance. Our findings support the hypothesis that variable or poikilotrophic environments promote diversification, and in particular, select for variation in ecotype more than phylotype.