Cyanobacterial Diversity and Halotolerance in a Variable Hypersaline Environment

The Great Salt Plains (GSP) in north-central Oklahoma, USA is an expansive salt flat (∼65 km²) 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.     

Aerobic biodegradation of benzene and toluene under hypersaline conditions at the Great Salt Plains, Oklahoma

The Great Salt Plains is a 65-km(2) hypersaline habitat of geological origin located in north-central Oklahoma. Contamination of such ecosystems by petroleum compounds is expected from non-point sources and due to increased human activities. Little information exists about the ability of halophilic and halotolerant bacteria present in such ancient and uncontaminated environments to degrade aromatic hydrocarbons. An enrichment culture was established from soil samples obtained from the salt flats using benzene as the sole carbon and energy source. The enrichment degraded benzene at varied salt concentrations ranging from 0 to 4M. Studies showed that roughly 33% of the (14)C-benzene was converted to (14)CO(2), indicating the mineralization capacity of native bacteria. Bacterial community structure analysis using denaturing gradient gel electrophoresis showed that different phylotypes were dominant at different salt concentrations.     

Halotolerant Aerobic Heterotrophic Bacteria from the Great Salt Plains of Oklahoma

The Salt Plains National Wildlife Refuge (SPNWR) near Cherokee, Oklahoma, contains a barren salt flat where Permian brine rises to the surface and evaporates under dry conditions to leave a crust of white salt. Rainfall events dissolve the salt crust and create ephemeral streams and ponds. The rapidly changing salinity and high surface temperatures, salinity, and UV exposure make this an extreme environment. The Salt Plains Microbial Observatory (SPMO) examined the soil microbial community of this habitat using classic enrichment and isolation techniques and phylogenetic rDNA studies. Rich growth media have been emphasized that differ in total salt concentration and composition. Aerobic heterotrophic enrichments were performed under a variety of conditions. Heterotrophic enrichments and dilution plates have generated 105 bacterial isolates, representing 46 phylotypes. The bacterial isolates have been characterized phenotypically and subjected to rDNA sequencing and phylogenetic analyses. Fast-growing isolates obtained from enrichments with 10% salt are predominantly from the gamma subgroup of the Proteobacteria and from the low GC Gram-positive cluster. Several different areas on the salt flats have yielded a variety of isolates from the Gram-negative genera Halomonas, Idiomarina, Salinivibrio, and Bacteroidetes. Gram-positive bacteria are well represented in the culture collection including members of the Bacillus, Salibacillus, Oceanobacillus, and Halobacillus.     

ALGAL COMMUNITY DYNAMICS AND HALOTOLERANCE IN A TERRESTRIAL,HYPERSALINE ENVIRONMENT1

We studied the algal community of the Great Salt Plains (GSP), an expansive (65 km²) salt flat situated in north‐central Oklahoma, USA that has been designated as the Salt Plains Microbial Observatory (SPMO) by the National Science Foundation. The GSP offered a unique opportunity to study a terrestrial, hypersaline algal community that experiences wide‐ranging environmental conditions. We were able to show that ammonium‐N, rather than salinity, was the most important predictor of total algal biomass. However, salinity was found to be a significant controlling variable in diatom distribution at the GSP, where diatom abundance was negatively correlated with porewater salinity concentrations. Overall, chlorophytes (likely dominated by Dunaliella spp.) were the most abundant algal group at the consistently hypersaline (>300 ppt) south site. Diatom and cyanobacterial biomass were on average highest at the central site, which experienced greater fluctuations in salinity. While taxonomic diversity was limited to three algal groups (chlorophytes, diatoms, and cyanobacteria), the salinity preferences and halotolerance ranges of isolated strains were quite variable. Although porewater salinities at the GSP are commonly near saturation (>300 ppt), the large majority of isolates had halotolerance ranges below 150 ppt. This suggests that algae at the GSP rarely achieve maximum growth rates, and could only do so when intermittent rain events reduce salinity to optimal levels. Because the vast majority of our strains were isolated from salt‐saturated soil samples, maintaining viability (rather than growth efficiency) appears to be the most successful adaptation to the extreme conditions at the GSP.     

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.     

Numerical taxonomy of heavy metal-tolerant nonhalophilic bacteria isolated from hypersaline environments.

A total of 232 metal-tolerant bacterial strains were isolated from water and sediment samples collected in different hypersaline environments located in Cádiz, Huelva and Morón de la Frontera (Spain). They were isolated on a medium containing mercury, chromium, cadmium, copper or zinc. These halotolerant isolates were analyzed by numerical taxonomy techniques by using the simple matching (SSM) and Jaccard (SJ) coefficients; clustering was achieved using the unweighted pair group method with averages (UPGMA) algorithm. At the 81% and 83% similarity level, different numbers of phenons were obtained for Gram-negative and Gram-positive halotolerant microorganisms. Most of the 48 Gram-negative metal-tolerant strains studied were grouped into nine phenons, representing the genera Pseudoalteromonas, Alteromonas, Xanthomonas, Pseudomonas, Alcaligenes and Enterobacteria. The 72 Gram-positive metal-tolerant strains grouped into eight phenons, with only 15 strains left unassigned. Most of the isolates were assigned to the genus Bacillus (seven phenons), and one phenon comprised microorganisms with phenotypic characteristics similar to those of the genus Celullomonas.     

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 km²) 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.     

Taxonomic analysis of extremely halophilic archaea isolated from 56-years-old dead sea brine samples

A taxonomic study comprising both phenotypic and genotypic characterization, has been carried out on a total of 158 extremely halophilic aerobic archaeal strains. These strains were isolated from enrichments prepared from Dead Sea water samples dating from 1936 that were collected by B. E. Volcani for the demonstration of microbial life in the Dead Sea. The isolates were examined for 126 morphological, physiological, biochemical and nutritional tests. Numerical analysis of the data, by using the S(J) coefficient and UPGMA clustering method, showed that the isolates clustered into six phenons. Twenty-two out of the 158 strains used in this study were characterized previously (ARAHAL et al., 1996) and were placed into five phenotypic groups. The genotypic study included both the determination of the guanineplus-cytosine content of the DNA and DNA-DNA hybridization studies. For this purpose, representative strains from the six phenons were chosen. These groups were found to represent some members of three different genera - Haloarcula (phenons A, B, and C), Haloferax (phenons D and E) and Halobacterium (phenon F) - of the family Halobacteriaceae, some of them never reported to occur in the Dead Sea, such as Haloarcula hispanica, while Haloferax volcanii (phenons D and E) was described in the Dead Sea by studies carried out several decades later than Volcani's work.     

Hydrocarbon biodegradation in hypersaline environments

When mineral oil, hexadecane, and glutamate were added to natural samples of varying salinity (3.3 to 28.4%) from salt evaporation ponds and Great Salt Lake, Utah, rates of metabolism of these compounds decreased as salinity increased. Rate limitations did not appear to relate to low oxygen levels or to the availability of organic nutrients. Some oxidation of l-[U-C]glutamic acid occurred even at extreme salinities, whereas oxidation of [1-C]hexadecane was too low to be detected. Gas chromatographic examination of hexane-soluble components of tar samples from natural seeps at Rozel Point in Great Salt Lake demonstrated no evidence of biological oxidation of isoprenoid alkanes subject to degradation in normal environments. Some hexane-soluble components of the same tar were altered by incubation in a low-salinity enrichment culture inoculated with garden soil. Attempts to enrich for microorganisms in saline waters able to use mineral oil as a sole source of carbon and energy were successful below, but not above, about 20% salinity. This study strongly suggests a general reduction of metabolic rate at extreme salinities and raises doubt about the biodegradation of hydrocarbons in hypersaline environments.     

Chemical and physical properties of some saline lakes in Alberta and Saskatchewan

Background

The Northern Great Plains of Canada are home to numerous permanent and ephemeral athalassohaline lakes. These lakes display a wide range of ion compositions, salinities, stratification patterns, and ecosystems. Many of these lakes are ecologically and economically significant to the Great Plains Region. A survey of the physical characteristics and chemistry of 19 lakes was carried out to assess their suitability for testing new tools for determining past salinity from the sediment record.

Results

Data on total dissolved solids (TDS), specific conductivity, temperature, dissolved oxygen (DO), and pH were measured in June, 2007. A comparison of these data with past measurements indicates that salinity is declining at Little Manitou and Big Quill Lakes in the province of Saskatchewan. However salinity is rising at other lakes in the region, including Redberry and Manito Lakes.

Conclusion

The wide range of salinities found across a small geographic area makes the Canadian saline lakes region ideal for testing salinity proxies. A nonlinear increase in salinity at Redberry Lake is likely influenced by its morphometry. This acceleration has ecological implications for the migratory bird species found within the Redberry Important Bird Area.     

Allozymatic variation in crustacea Artemia franciscana (Anostraca: Artemiidae) of Freat Salt Lake in different experimental conditions

Starch gel electrophoresis was used to analyze the allelic variability of four polymorphic loci (Lap-2, Lap-3, Pgm and Gpi) from a single population of Artemia franciscana (Kellogg, 1906) from the Great Salt Lake (Utah, USA), cultured under eight different experimental conditions. The organisms were cultured to the adult stage under a 2 x 2 x 2 experimental design (22 and 30 degrees C; 30 and 60 ppt salinity; and Dunaliella sp. and Spirulina sp. as food). There were significant differences in allele frequencies at each locus and the mean expected heterozygosity (He) varied from 0.236 to 0.447. Therefore, the hypothesis of no allelic differences among treatments is rejected. With relation to a possible correlation between genetic variability and the phenotypic characteristics, the results show that there is probably a synergic effect between the different salinities and temperatures on the survival of heterozygous organisms in the different loci.     

DNA-Repair Potential of <Emphasis Type="Italic">Halomonas</Emphasis> spp. from the Salt Plains Microbial Observatory of Oklahoma

The Great Salt Plains (GSP), an unvegetated, barren salt flat that is part of the Salt Plains National Wildlife Refuge near Cherokee, Oklahoma, is the site of the Salt Plains Microbial Observatory. At the GSP the briny remains of an ancient sea rise to the surface, evaporate under dry conditions, and leave crusts of white salt. Adaptation to this environment requires development of coping mechanisms providing tolerance to desiccating conditions due to the high salinity, extreme temperatures, alkaline pH, unrelenting exposure to solar UV radiation, and prevailing winds. Several lines of evidence suggest that the same DNA repair mechanisms that are usually associated with UV light or chemically induced DNA damage are also important in protecting microbes from desiccation. Because little is known about the DNA repair capacity of microorganisms from hypersaline terrestrial environments, we explored the DNA repair capacity of microbial isolates from the GSP. We used survival following exposure to UV light as a convenient tool to assess DNA repair capacity. Two species of Halomonas (H. salina and H. venusta) that have been isolated repeatedly from the GSP were chosen for analysis. The survival profiles were compared to those of Escherichia coli, Pseudomonas aeruginosa, and Halomonas spp. from aquatic saline environments. Survival of GSP organisms exceeded that of the freshwater organism P. aeruginosa, although they survived no better than E. coli. The GSP isolates were much more resistance to killing by UV than were the aquatic species of Halomonas reported in the literature [Martin et al. (2000) Can J Microbiol 46:180–187]. Unlike E. coli, the GSP isolates did not appear to have an inducible, error-prone repair mechanism. However, they demonstrated high levels of spontaneous mutation.     

Occurrence and characterization of psychrotolerant hydrocarbon-oxidizing bacteria from surface seawater along the Victoria Land coast (Antarctica)

A total of 253 hydrocarbon-oxidizing bacterial isolates were achieved from eight Antarctic surface seawater samples enriched on diesel oil at 4°C. Isolates were screened by amplified ribosomal DNA restriction analysis prior to 16S rRNA gene sequencing. Sequences were compared to those in available databases using the Basic Local Alignment Search Tool network service to determine their approximate phylogenetic affiliations. The majority of the isolates were affiliated to the Actinobacteria (75.9%) and the Gamma-Proteobacteria (22.9%). The Alpha- and Beta-Proteobacteria represented 0.8 and 0.4% of total isolates, respectively. The Actinobacteria were predominantly allocated to the genera Arthrobacter, Cryobacterium and Rhodococcus. The Gamma-Proteobacteria were mainly found to be related to the genus Pseudomonas. Conversely, the Alpha- and Beta-Proteobacterial isolates shared the highest degree of sequence identity with unclassified bacteria. Differences in the distribution of the detected phylotypes were observed among the analyzed samples. Isolates representing each phylotype were selected for further characterization, including phenotypic assays and screening for the growth ability in the presence of individual hydrocarburic substrates as the sole supplied carbon and energy source. Isolates possessed different patterns of substrate utilization. Aliphatic hydrocarbons supported the growth of a higher number of isolates than aromatics. Results confirm the ability of our Antarctic marine bacteria to utilize hydrocarbons at low temperature and therefore suggesting that isolates with different substrate specificities can act in nature as a consortium in the utilization of complex hydrocarburic mixtures.     

Halophilic-blue-green algae

The isolation of a halophilic blue-green alga, Aphanothece halophytica, from Great Salt Lake is described. The organism was cultured from waters with salinities up to saturated NaCl (about 30% w/v). It has an optimum salinity for growth of about 16% NaCl, but can grow very slowly even in saturated NaCl. Based on the study of the Great Salt Lake organism, and on a review of the earlier literature, it is concluded that despite recent reports to the contrary, true halophilic blue-green algae do exist.     

Novel green sulfur bacteria phylotypes detected in saline environments: ecophysiological characters versus phylogenetic taxonomy

The taxonomic significance of salt tolerance or requirements in green sulfur bacteria has been analyzed with environmental populations and enrichment cultures from several saline systems (inland and coastal water bodies) with different salinities (salt composition and concentration). Novel phylotypes of green sulfur bacteria have been found in hypersaline and brackish environments and 16S rRNA gene sequence analysis affiliated them into phylogenetic groups in which neither halotolerant nor halophilic species have been known to date. Therefore, salt tolerance does not seem to be restricted to members of any specific subgroup but is widespread among all the different phylogenetic branches of the green sulfur bacteria group, and closely-related phylotypes can have dissimilar salt tolerance capacities. Thus the phenotypic characteristics and phylogenetic structure of the green sulfur bacteria present some incongruities. Phenotypic traits should be studied further in order to determine the ecophysiological features of green sulfur bacteria phylotypes.     

Land use patterns,ecoregion, and microcystin relationships in U.S. lakes and reservoirs: A preliminary evaluation

A statistically significant association was found between the concentration of total microcystin, a common class of cyanotoxins, in surface waters of lakes and reservoirs in the continental U.S. with watershed land use using data from 1156 water bodies sampled between May and October 2007 as part of the USEPA National Lakes Assessment. Nearly two thirds (65.8%) of the samples with microcystin concentrations ≥1.0 μg/L (n = 126) were limited to three nutrient and water quality-based ecoregions (Corn Belt and Northern Great Plains, Mostly Glaciated Dairy Region, South Central Cultivated Great Plains) in watersheds with strong agricultural influence. canonical correlation analysis (CCA) indicated that both microcystin concentrations and cyanobacteria abundance were positively correlated with total nitrogen, dissolved organic carbon, and temperature; correlations with total phosphorus and water clarity were not as strong. This study supports a number of regional lake studies that suggest that land use practices are related to cyanobacteria abundance, and extends the potential impacts of agricultural land use in watersheds to include the production of cyanotoxins in lakes.     

Genotypic and phenotypic diversity of phlD-containing Pseudomonas strains isolated from the rhizosphere of wheat

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent Pseudomonas strains containing phlD, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2, 4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.     

Diversity within cyanobacterial mat communities in variable salinity meltwater ponds of McMurdo Ice Shelf, Antarctica

This study investigated the diversity of cyanobacterial mat communities of three meltwater ponds--Fresh, Orange and Salt Ponds, south of Bratina Island, McMurdo Ice Shelf, Antarctica. A combined morphological and genetic approach using clone libraries was used to investigate the influence of salinity on cyanobacterial diversity within these ecosystems without prior cultivation or isolation of cyanobacteria. We were able to identify 22 phylotypes belonging to Phormidium sp., Oscillatoria sp. and Lyngbya sp. In addition, we identified Antarctic Nostoc sp., Nodularia sp. and Anabaena sp. from the clone libraries. Fresh (17 phylotypes) and Orange (nine phylotypes) Ponds showed a similar diversity in contrast to that of the hypersaline Salt Pond (five phylotypes), where the diversity within cyanobacterial mats was reduced. Using the comparison of identified phylotypes with existing Antarctic sequence data, it was possible to gain further insight into the different levels of distribution of phylotypes identified in the investigated cyanobacterial mat communities of McMurdo Ice Shelf.     

Hemolymph patterns of free amino acids in the brine shrimp Artemia franciscana after three days starvation at different salinities

The hemolymph pattern of free amino acids was examined in the brine shrimp, Artemia franciscana (Great Salt Lake origin). After one-month acclimation to 35 or 60 ppt salinity at 27 degrees C, the animals were transferred to 10, 35 or 60 ppt salinities to continue acclimation for 3 days without feeding at 27 degrees C. The osmolarity of one of the new media was raised by glycerol addition. In the hemolymph, 8 amino acids such as taurine, alanine, threonine, serine, lysine, glycine, arginine and leucine, comprised approximately 70% of the total content of free amino acids. This pattern suggested internal proteolysis due to starvation at high temperature. The total content of free amino acids significantly increased at 10 and 60 ppt salinities in comparison to 35 ppt. The hemolymph patterns from the 10 ppt and glycerol-added media showed a singularly high peak of taurine or alanine.     

A molecular systematic survey of cultured microbial associates of deep-water marine invertebrates

A taxonomic survey was conducted to determine the microbial diversity held within the Harbor Branch Oceanographic Marine Microbial Culture Collection (HBMMCC). The collection consists of approximately 17,000 microbial isolates, with 11,000 from a depth of greater than 150 ft seawater. A total of 2273 heterotrophic bacterial isolates were inventoried using the DNA fingerprinting technique amplified rDNA restriction analysis on approximately 750-800 base pairs (bp) encompassing hypervariable regions in the 5' portion of the small subunit (SSU) 16S rRNA gene. Restriction fragment length polymorphism patterns obtained from restriction digests with RsaI, HaeIII, and HhaI were used to infer taxonomic similarity. SSU 16S rDNA fragments were sequenced from a total of 356 isolates for more definitive taxonomic analysis. Sequence results show that this subset of the HBMMCC contains 224 different phylotypes from six major bacterial clades (Proteobacteria (Alpha, Beta, Gamma), Cytophaga, Flavobacteria, and Bacteroides (CFB), Gram + high GC content, Gram + low GC content). The 2273 microorganisms surveyed encompass 834 alpha-Proteobacteria (representing 60 different phylotypes), 25 beta-Proteobacteria (3 phylotypes), 767 gamma-Proteobacteria (77 phylotypes), 122 CFB (17 phylotypes), 327 Gram + high GC content (43 phylotypes), and 198 Gram + low GC content isolates (24 phylotypes). Notably, 11 phylotypes were < or =93% similar to the closest sequence match in the GenBank database even after sequencing a larger portion of the 16S rRNA gene (approximately 1400 bp), indicating the likely discovery of novel microbial taxa. Furthermore, previously reported "uncultured" microbes, such as sponge-specific isolates, are part of the HBMMCC. The results of this research will be available online as a searchable taxonomic database (www.hboi.edu/dbmr/dbmr_hbmmd.html).