Unveiling the Gut Microbiota and Resistome of Wild Cotton Mice, Peromyscus gossypinus, from Heavy Metal- and Radionuclide-Contaminated Sites in the Southeastern United States.

The prevalence of antibiotic resistance genes (ARGs) can be driven by direct selection from antibiotic use and indirect selection from substances such as heavy metals (HMs). While significant progress has been made to characterize the influence of HMs on the enrichment and dissemination of ARGs in the environment, there is still much we do not know. To fill this knowledge gap, we present a comprehensive analysis of gut bacteria associated with wild cotton mice (Peromyscus gossypinus) trapped from several areas affected by legacies of HM and radionuclide contamination. We explore how these contaminants affect gut microbial community (GMC) composition and diversity and the enrichment of antibiotic, biocide, and metal resistance genes. Although we were able to identify that a myriad of co-occurring antimicrobial and HM resistance genes appear in mice from all areas, including those without a history of contamination, the proportions of co-occurring ARGs and metal resistance genes (MRGs) are higher in sites with radionuclide contamination. These results support those from several previous studies and enhance our understanding of the coselection process, while providing new insights into the ubiquity of antimicrobial resistance in the resistome of wild animals. IMPORTANCE Antimicrobial resistance is a serious global public health concern because of its prevalence and ubiquitous distribution. The rapid dissemination of antibiotic resistance genes is thought to be the result of the massive overuse of antibiotics in agriculture and therapeutics. However, previous studies have demonstrated that the spread of antibiotic resistance genes can also be influenced by heavy metal contamination. This coselection phenomenon, whereby different resistance determinants are genetically linked on the same genetic element (coresistance) or a single genetic element provides resistance to multiple antimicrobial agents (cross-resistance), has profound clinical and environmental implications. In contrast to antibiotics, heavy metals can persist in the environment as a selection pressure for long periods of time. Thus, it is important to understand how antibiotic resistance genes are distributed in the environment and to what extent heavy metal contaminants may be driving their selection, which we have done in one environmental setting.

Retraction Note: Molecular Phylogeny and Revision of Copepod Orders (Crustacea: Copepoda).

Editor's Note: this Article has been retracted; the Retraction Note is available at https://www.nature.com/articles/s41598-020-72330-x.

Metal accumulation in dragonfly nymphs and crayfish as indicators of constructed wetland effectiveness.

Constructed wetland effectiveness is often assessed by measuring reductions of contaminant concentrations in influent versus departing effluent, but this can be complicated by fluctuations in contaminant content/chemistry and hydrology. We assessed effectiveness of a constructed wetland at protecting downstream biota from accumulating elevated metal concentrations-particularly copper and zinc in effluents from a nuclear materials processing facility. Contaminants distributed throughout a constructed wetland system and two reference wetlands were assessed using six dragonfly nymph genera (Anax, Erythemis, Libellula, Pachydiplax, Tramea, and Plathemis) as biomonitors. Additionally, the crayfish, Cambarus latimanus, were analyzed from the receiving and two reference streams. Concentrations of Cu, Zn, Pb, Mn, Cr, Cd, and Al were evaluated in 597 dragonfly nymph and 149 crayfish whole-body composite samples. Dragonfly genera varied substantially in metal accumulation and the ability to identify elevated metal levels throughout components of the constructed wetland. Genera more closely associated with bottom sediments tended to accumulate higher levels of metals with Libellula, Pachydiplax, and Erythemis often accumulating highest concentrations and differing most among sites. This, combined with their abundance and broad distributions make the latter two species suitable candidates as biomonitors for constructed wetlands. As expected, dragonfly nymphs accumulated higher metal concentrations in the constructed wetland than reference sites. However, dragonfly nymphs often accumulated as high of metal concentrations downstream as upstream of the water treatment cells. Moreover, crayfish from the receiving stream near the constructed wetland accumulated substantially higher Cu concentrations than from downstream locations or reference streams. Despite reducing metal concentrations at base flow and maintaining regulatory compliance, metal fluxes from the wetland were sufficient to increase accumulation in downstream biota. Future work should evaluate the causes of downstream accumulation as the next step necessary to develop plans to improve the metal sequestering efficiency of the wetland under variable flow regimes.

Basins, beaver ponds, and the storage and redistribution of trace elements in an industrially impacted coastal plain stream on the Savannah River Site, SC, USA.

Accumulation of eleven trace elements in sediment was evaluated throughout an industrially disturbed headwater stream on the Savannah River Site, SC, USA. Sampling began at upstream sedimentation basins at the margins of industrial areas, continued longitudinally downstream to a beaver pond representing a potential sink in the mid-reaches, and ended in downstream reaches. Additionally, sediment from beaver impacted areas in another industrially disturbed stream and a reference stream were analyzed to assess the natural tendency of these depositional features to settle out trace elements. We further compared trace element accumulation in sediment and biota from downstream reaches before and after an extreme rainy period to evaluate the potential redistribution of trace elements from sink areas. Trace elements accumulated in the headwater basins from which elements were redistributed to downstream reaches. The mid-reach beaver affected area sediments accumulated elevated concentrations of most analyzed elements compared to the free-flowing stream. The elevated accumulation of organic matter in these sink areas illustrated the effectiveness of reduced water velocity areas to settle out materials. The natural tendency of beaver ponds to accumulate trace elements and organic matter was further illustrated by sediments from the reference beaver pond accumulating higher concentrations of several elements than sediments from the free flowing section the stream impacted by industrial activity. However, concentrations in sediment from sedimentation basins and the beaver impacted area of the disturbed stream were highest. Trace elements and organic matter appeared to be redistributed from the sinks after the record rainy period resulting in increased trace element concentrations in both sediment and biota. These data suggest that assessments of contaminants in stream systems should include such slow-water, extreme depositional zones such as beaver impacted areas or basins to verify what contaminants may be pulsing through the stream.

Sediment and biota trace element distribution in streams disturbed by upland industrial activity.

Extensive industrial areas in headwater stream watersheds can severely impact the physical condition of streams and introduce contaminants. We compared 3 streams that received stormwater runoff and industrial effluents from industrial complexes to 2 reference streams. Reference streams provide a benchmark of comparison of geomorphic form and stability in coastal plain, sandy-bottomed streams as well as concentrations of trace elements in sediment and biota in the absence of industrial disturbance. We used crayfish (Cambarus latimanus, Procambarus raneyi, Procambarus acutus) and crane fly larvae (Tipula) as biomonitors of 15 trace elements entering aquatic food webs. Streams with industrial areas were more scoured, deeply incised, and less stable. Sediment organic matter content broadly correlated to trace element accumulation, but fine sediments and organic matter were scoured from the bottoms of disturbed streams. Trace element concentrations were higher in depositional zones than runs within all streams. Despite contaminant sources in the headwaters, trace element concentrations were generally not elevated in sediments of the eroded streams. However, element concentrations were frequently elevated in biota from these streams with taxonomic differences in accumulation amplified. In eroded, sand-bottomed coastal plain streams with unstable sediments, single snapshots of sediment trace element concentrations did not characterize well bioavailable trace elements. Biota that integrated exposures over time and space within their home ranges better detected bioavailable contaminants than sediment. Environ Toxicol Chem 2019;38:115-131. © 2018 SETAC.

Molecular Phylogeny and Revision of Copepod Orders (Crustacea: Copepoda).

For the first time, the phylogenetic relationships between representatives of all 10 copepod orders have been investigated using 28S and 18S rRNA, Histone H3 protein and COI mtDNA. The monophyly of Copepoda (including Platycopioida Fosshagen, 1985) is demonstrated for the first time using molecular data. Maxillopoda is rejected, as it is a polyphyletic group. The monophyly of the major subgroups of Copepoda, including Progymnoplea Lang, 1948 (=Platycopioida); Neocopepoda Huys and Boxshall, 1991; Gymnoplea Giesbrecht, 1892 (=Calanoida Sars, 1903); and Podoplea Giesbrecht, 1892, are supported in this study. Seven copepod orders are monophyletic, including Platycopioida, Calanoida, Misophrioida Gurney, 1933; Monstrilloida Sars, 1901; Siphonostomatoida Burmeister, 1834; Gelyelloida Huys, 1988; and Mormonilloida Boxshall, 1979. Misophrioida (=Propodoplea Lang, 1948) is the most basal Podoplean order. The order Cyclopoida Burmeister, 1835, is paraphyletic and now encompasses Poecilostomatoida Thorell, 1859, as a sister to the family Schminkepinellidae Martinez Arbizu, 2006. Within Harpacticoida Sars, 1903, both sections, Polyarthra Lang, 1948, and Oligoarthra Lang, 1948, are monophyletic, but not sister groups. The order Canuelloida is proposed while maintaining the order Harpacticoida s. str. (Oligoarthra). Cyclopoida, Harpacticoida and Cyclopinidae are redefined, while Canuelloida ordo. nov., Smirnovipinidae fam. nov. and Cyclopicinidae fam. nov are proposed as new taxa.

The effects of low-level ionizing radiation and copper exposure on the incidence of antibiotic resistance in lentic biofilm bacteria.

Environmental reservoirs of antibiotic resistant bacteria are poorly understood. Understanding how the environment selects for resistance traits in the absence of antibiotics is critical in developing strategies to mitigate this growing menace. Indirect or co-selection of resistance by environmental pollution has been shown to increase antibiotic resistance. However no attention has been given to the effects of low-level ionizing radiation or the interactions between radiation and heavy metals on the maintenance or selection for antibiotic resistance (AR) traits. Here we explore the effect of radiation and copper on antibiotic resistance. Bacteria were collected from biofilms in two ponds - one impacted by low-level radiocesium and the other an abandoned farm pond. Through laboratory controlled experiments we examined the effects of increasing concentrations of copper on the incidence of antibiotic resistance. Differences were detected in the resistance profiles of the controls from each pond. Low levels (0.01 mM) of copper sulfate increased resistance but 0.5 mM concentrations of copper sulfate depressed the AR response in both ponds. A similar pattern was observed for levels of multiple antibiotic resistance per isolate. The first principal component response of isolate exposure to multiple antibiotics showed significant differences among the six isolate treatment combinations. These differences were clearly visualized through a discriminant function analysis, which showed distinct antibiotic resistance response patterns based on the six treatment groups.

Trace element accumulation in lotic dragonfly nymphs: Genus matters.

Constituents of coal combustion waste (CCW) expose aquatic organisms to complex mixtures of potentially toxic metals and metalloids. Multi-element trace element analyses were used to distinguish patterns of accumulation among 8 genera of dragonfly nymphs collected from two sites on a CCW contaminated coastal plain stream. Dragonfly nymphs are exceptional for comparing trace element accumulation in syntopic macroinvertebrates that are all predators within the same order (Odonata) and suborder (Anisoptera), but differ vastly in habitat use and body form. Sixteen trace element (Be, V, Cr, Ni, Cu, Zn, As, Se, Sr, Cd, Sb, Cs, Ba, Hg, Tl, and Pb) were analyzed and trophic position and basal carbon sources assessed with stable isotope analyses (C and N). Trophic positions varied within relatively narrow ranges. Size did not appear to influence trophic position. Trophic position rarely influenced trace element accumulation within genera and did not consistently correlate with accumulation among genera. Patterns between δ13C and trace element accumulation were generally driven by differences between sites. An increase in trace element accumulation was associated with a divergence of carbon sources between sites in two genera. Higher trace element concentrations tended to accumulate in nymphs from the upstream site, closer to contaminant sources. Influences of factors such as body form and habitat use appeared more influential on trace element accumulation than phylogeny for several elements (Ni, Ba, Sr, V, Be, Cd, and Cr) as higher concentrations accumulated in sprawler and the climber-sprawler genera, irrespective of family. In contrast, As and Se accumulated variably higher in burrowers, but accumulation in sprawlers differed between sites. Greater variation between genera than within genera suggests genus as an acceptable unit of comparison in dragonfly nymphs. Overall, taxonomic differences in trace element accumulation can be substantial, often exceeding variation between sites. Our results underscore the element and taxa specific nature of trace element accumulation, but we provide evidence of accumulation of some trace elements differing among dragonflies that differ in body form and utilize different sub-habitats within a stream reach.

Exposure of juvenile Leghorn chickens to lead acetate enhances antibiotic resistance in enteric bacterial flora.

Heavy metals have been implicated for their ability to increase antibiotic resistance in bacteria collected from polluted waters, independent of antibiotic exposure. Specific-pathogen-free Leghorn chickens were therefore given Pb acetate in the drinking water to expose the enteric bacteria to Pb and to determine if antibiotic resistance changed in these bacteria. Concentrations of Pb used were 0.0, 0.01, 0.1, 1.0, or 10.0 mM; birds given the highest 2 concentrations showed signs of moribundity and dehydration and were removed from the study. Vent culture samples were collected for bacterial cultures on d 0 before Pb exposure, d 7 and 14, and then birds were euthanized by CO2 gas for necropsy on d 14, at which time intestinal contents were also collected for bacterial cultures. Fecal swabs but not intestinal samples from Pb-exposed birds contained isolates that had significantly elevated antibiotic resistance. Some of the isolates contained bacteria that were resistant to up to 20 antibiotics. These results suggest the need for repeated studies in chickens infected with zoonotic pathogens.

Variation in trace-element accumulation in predatory fishes from a stream contaminated by coal combustion waste.

Extensive and critical evaluation can be required to assess contaminant bioaccumulation in large predatory fishes. Species differences in habitat use, resource use, and trophic level, often influenced by body form, can result in diverging contaminant bioaccumulation patterns. Moreover, the broad size ranges inherent with large-bodied fish provide opportunity for trophic and habitat shifts within species that can further influence contaminant exposure. We compared contaminant bioaccumulation in four fish species, as well as two herbivorous invertebrates, from a coal combustion waste contaminated stream. Muscle, liver, and gonad tissue were analyzed from fish stratified across the broadest size ranges available. Effects of trophic position (δ (15)N), carbon sources (δ (13)C), and body size varied among and within species. Mercury and cesium concentrations were lowest in the invertebrates and increased with trophic level both among and within fish species. Other elements, such as vanadium, cadmium, barium, nickel, and lead, had greater levels in herbivorous invertebrates than in fish muscle. Sequestration by the fish livers averted accumulation in muscle. Consequently, fish liver tissue appeared to be a more sensitive indicator of bioavailability, but exceptions existed. Despite liver sequestration, within fishes, muscle concentrations of many elements still tended to increase by trophic level. Notable variation within some species was observed. These results illustrate the utility of stable isotope data in exploring differences of bioaccumulation within taxa. Our analyses suggest a need for further evaluation of the underlying sources of this variability to better understand contaminant bioaccumulation in large predatory fishes.

Antimicrobial textiles.

Bacteria have evolved unique mechanisms that allow them survive in the presence of strong selection pressures. Included in these mechanisms is the ability to share genetic determinants among and between species of bacteria thus spreading metal or antibiotic resistance traits quickly. The textile industry in response to demand has developed antimicrobial fabrics by the addition of bactericidal compounds during production. Some of these antimicrobials include metal nanoparticles, quaternary ammonia compounds, and broad spectrum compounds like triclosan. Bacteria have already expressed resistance to each of these bactericides. Here we discuss the evolutionary and ecological consequences of antimicrobial textiles in terms of co-selection. We predict that continued use of such materials could result in increased and widespread resistance to specific antimicrobials, especially metals, with an increased resistance to antibiotics. Such increases have the potential to find their way into other bacterial populations of human pathogens leading to serious and unintended public health consequences.

Rapid in situ detection of virulent Vibrio vulnificus strains in raw oyster matrices using real-time PCR.

Vibrio vulnificus is a Gram-negative bacterial pathogen responsible for the vast majority of bacterially mediated fatalities from the consumption of raw or undercooked seafood in the USA. Vibrio vulnificus-associated septicaemia can occur rapidly (< 24 h); however, methods for the isolation and confirmation of V. vulnificus from seafood samples typically require several days. A real-time PCR assay was developed for V. vulnificus biotype 1 that provides a rapid means of identifying a gene fragment (vcgC) previously indicated as a strong predictor of potential virulence. PCR probe specificity was confirmed by amplification of 17 clinical V. vulnificus strains and by the lack of amplification with seven non-pathogenic V. vulnificus isolates and a wide range of closely related bacteria. Oyster and seawater samples were amended with a range of environmentally realistic concentrations of C-genotype V. vulnificus cells, which were quantitatively and unambiguously identified according to biotype. Of some significance, we utilized a sample processing and nucleic acid extraction procedure that allowed identification of pathogenic strains of V. vulnificus from oyster matrices without prior enrichment or culturing of strains. This outlined approach allowed the detection of as little as 50 cfu of V. vulnificus in less than 5 h, which compares favourably with culture-based approaches. The results indicate the applicability of this approach for monitoring purposes or as a potential diagnostic tool in clinical settings.

Influence of industrial contamination on mobile genetic elements: class 1 integron abundance and gene cassette structure in aquatic bacterial communities.

The acquisition of new genetic material via horizontal gene transfer allows bacteria to rapidly evolve. One key to estimating the contribution of horizontal gene transfer to bacterial evolution is to quantify the abundance of mobile genetic elements (MGEs) in bacterial communities under varying degrees of selective pressure. We quantified class 1 integrase (intI1) gene abundance in total community DNA extracted from contaminated and reference riverine and estuarine microhabitats, and in metal- or antibiotic-amended freshwater microcosms. The intI1 gene was more abundant in all contaminant-exposed communities indicating that relative gene transfer potential is higher in these communities. A second key to assessing the contributions of MGEs to bacterial evolution is to examine the structure and function of the MGE-associated gene pool. We determined that the gene cassette pool is a novel and diverse resource available for bacterial acquisition, but that contamination has no discernible effect on cassette richness. Gene cassette profiles were more similar within sites than among sites, yet bacterial community profiles were not, suggesting that selective pressures can shape the structure of the gene cassette pool. Of the 46 sequenced gene cassette products, 37 were novel sequences, while the 9 gene cassettes with similarity to database sequences were primarily to hypothetical proteins. That class 1 integrons are ubiquitous and abundant in environmental bacterial communities indicates that this group of MGEs can play a substantial role in the acquisition of a diverse array of gene cassettes beyond their demonstrated impact in mediating multidrug resistance in clinical bacteria.

Spatial analysis of antibiotic resistance along metal contaminated streams.

The spatial pattern of antibiotic resistance in culturable sediment bacteria from four freshwater streams was examined. Previous research suggests that the prevalence of antibiotic resistance may increase in populations via indirect or coselection from heavy metal contamination. Sample bacteria from each stream were grown in media containing one of four antibiotics-tetracycline, chloramphenicol, kanamycin, and streptomycin-at concentrations greater than the minimum inhibitory concentration, plus a control. Bacteria showed high susceptibilities to the former two antibiotics. We summarized the latter two more prevalent (aminoglycoside) resistance responses and ten metals concentrations per sediment sample, by Principal Components Analysis. Respectively, 63 and 58% of the variability was explained in the first principal component of each variable set. We used these multivariate summary metrics [i.e., first principal component (PC) scores] as input measures for exploring the spatial correlation between antibiotic resistance and metal concentration for each stream sampled. Results show a significant and negative correlation between metals PC scores versus aminoglycoside resistance scores and suggest that selection for metal tolerance among sediment bacteria may influence selection for antibiotic resistance differently in sediments than in the water column. Our most important finding comes from geostatistical cross-variogram analysis, which shows that increasing metal concentration scores are spatially associated with decreasing aminoglycoside resistance scores--a negative correlation, but holds for contaminated streams only. We suspect our field results are influenced by metal bioavailability in the sediments and by a contaminant promoted interaction or "cocktail effect" from complex combinations of pollution mediated selection agents.

Novel tetracycline resistance determinant isolated from an environmental strain of Serratia marcescens.

Resistances to tetracycline and mercury were identified in an environmental strain of Serratia marcescens isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in Escherichia coli. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [tetA(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of Acinetobacter spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the tetA(41) gene was a divergently transcribed gene [tetR(41)] predicted to encode a tetracycline-responsive repressor protein. The tetA(41)-tetR(41) intergenic region contained putative operators for TetR(41) binding. The tetA(41) and tetR(41) promoters were analyzed using lacZ fusions, which showed that the expression of both the tetA(41) and tetR(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this S. marcescens strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the S. marcescens chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to E. coli, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.

Bacterial tolerances to metals and antibiotics in metal-contaminated and reference streams.

Anthropogenic-derived sources of selection are typically implicated as mechanisms for maintaining antibiotic resistance in the environment. Here we report an additional mechanism for maintaining antibiotic resistance in the environment through bacterial exposure to metals. Using a culture-independent approach, bacteria sampled along a gradient of metal contamination were more tolerant of antibiotics and metals compared to bacteria from a reference site. This evidence supports the hypothesis that metal contamination directly selects for metal tolerant bacteria while co-selecting for antibiotic tolerant bacteria. Additionally, to assess how antibiotic and metal tolerance may be transported through a stream network, we studied antibiotic and metal tolerance patterns over three months in bacteria collected from multiple stream microhabitats including the water column, biofilm, sediment and Corbicula fluminea (Asiatic clam) digestive tracts. Sediment bacteria were the most tolerant to antibiotics and metals, while bacteria from Corbicula were the least tolerant. Differences between microhabitats may be important for identifying reservoirs of resistance and for predicting how these genes are transferred and transported in metal-contaminated streams. Temporal dynamics were not directly correlated to a suite of physicochemical parameters, suggesting that tolerance patterns within microhabitats are linked to a complex interaction of the physicochemical characteristics of the stream.

Lipid composition of suspended particulate matter (SPM) in a southeastern blackwater stream.

Suspended particulate matter (SPM) was collected seasonally for 1 yr at third- and fifth-order sites in a blackwater stream on the coastal plain in South Carolina. Fatty acids with carbon chain lengths from C(12)-C(32) were the most abundant component among the lipid classes examined with total concentrations varying from 8.5 to 60.2 microgL(-1). Unsaturated fatty acids predominated while significant concentrations of the even-chained saturated components C(24)-C(30) derived from cuticular plant waxes were also found. Concentrations of aliphatic alcohols, with carbon chain lengths between C(16) and C(30), ranged from 0.52 to 2.73 microgL(-1) and was dominated by the higher molecular weight compounds (C(22)-C(30)) derived primarily from cuticular plant waxes. Total hydrocarbon concentrations ranged from 0.35 to 5.66 microgL(-1) and showed no discernible trends with time or consistent difference between sites. The hydrocarbon assemblage observed indicates that these components are entirely of biogenic origin with no detectable anthropogenic contribution. The ratios of unsaturated to saturated fatty acids and cuticular to noncuticular fatty acids and alcohols at both stream locations indicate that the organic detritus associated with SPM collected during November and January is of more recent origin and less processed than that collected in the spring and summer months. Lipid concentrations at the third-order site generally had higher and more variable concentrations of the lipid classes compared with the fifth-order site. The ratio of saturated to unsaturated fatty acids was higher at the third-order site indicating that the organic fraction of SPM at this site was of more recent origin and less decomposed. The ratios of cuticular to noncuticular fatty acids and alcohols support this conclusion. These results indicate an export of particulate lipids of higher carbon resource quality from upstream to lower stream reaches.

Recovery of novel bacterial diversity from a forested wetland impacted by reject coal.

Sulphide mineral mining together with improperly contained sulphur-rich coal represents a significant environmental problem caused by leaching of toxic material. The Savannah River Site's D-area harbours a 22-year-old exposed reject coal pile (RCP) from which acidic, metal rich, saline runoff has impacted an adjacent forested wetland. In order to assess the bacterial community composition of this region, composite sediment samples were collected at three points along a contamination gradient (high, middle and no contamination) and processed for generation of bacterial and archaeal 16S rDNA clone libraries. Little sequence overlap occurred between the contaminated (RCP samples) and unimpacted sites, indicating that the majority of 16S rDNAs retrieved from the former represent organisms selected by the acidic runoff. Archaeal diversity within the RCP samples consisted mainly of sequences related to the genus Thermoplasma and to sequences of a novel type. Bacterial RCP libraries contained 16S rRNA genes related to isolates (Acidiphilium sp., Acidobacterium capsulatum, Ferromicrobium acidophilium and Leptospirillum ferrooxidans) and environmental clones previously retrieved from acidic habitats, including ones phylogenetically associated with organisms capable of sulphur and iron metabolism. These libraries also exhibited particularly novel 16S rDNA types not retrieved from other acid mine drainage habitats, indicating that significant diversity remains to be detected in acid mine drainage-type systems.

Characterization of riparian species and stream detritus using multiple stable isotopes.

Multiple stable isotopes were used to determine the effectiveness of distinguishing among several dominant riparian species and aquatic macrophytes both spatially (three sites) and temporally (three seasons) along an 8-km reach of a blackwater stream. The differences in isotopic composition were used to assess contributions of various organic matter sources to the detrital pool of the stream. Samples of riparian and aquatic macrophyte vegetation and detritus were collected at three times to represent early leaf-out (April), mid-summer (August), and just prior to abscission (October). Each sample was analyzed for stable isotopes of carbon δ13C, nitrogen δ15N, and sulfur δ34S Within a site and sampling date, δ13C-values were significantly different among certain riparian species and detritus samples. Species differences persisted between seasons. δ34S values were the most variable of the three elements examined although they remained fairly constant through time within each species and site. The results suggest that temporal changes in isotopic compositions of riparian species and aquatic macrophytes are site-specific. Discriminant analysis dissimilarity plots (based on all three isotopes) demonstrated that the contribution of species to the detrital pool depended on the site and season. At the upper site, detritus was isotopically most similar to Quercus laurifolia and Sparganium americanum in April, and the aquatic macrophytes (S. americanum and Potamogeton spp.) in August and October. At the middle site, detritus was most similar to Carpinus caroliniana and Q. nigra in April but no single source was similar to detritus in August or October. At the lower site, detritus was most similar to Taxodium distichum for all three seasons.