Early episodes of high-pressure core formation preserved in plume mantle.

The decay of short-lived iodine (I) and plutonium (Pu) results in xenon (Xe) isotopic anomalies in the mantle that record Earth's earliest stages of formation. Xe isotopic anomalies have been linked to degassing during accretion, but degassing alone cannot account for the co-occurrence of Xe and tungsten (W) isotopic heterogeneity in plume-derived basalts and their long-term preservation in the mantle. Here we describe measurements of I partitioning between liquid Fe alloys and liquid silicates at high pressure and temperature and propose that Xe isotopic anomalies found in modern plume rocks (that is, rocks with elevated 3He/4He ratios) result from I/Pu fractionations during early, high-pressure episodes of core formation. Our measurements demonstrate that I becomes progressively more siderophile as pressure increases, so that portions of mantle that experienced high-pressure core formation will have large I/Pu depletions not related to volatility. These portions of mantle could be the source of Xe and W anomalies observed in modern plume-derived basalts. Portions of mantle involved in early high-pressure core formation would also be rich in FeO, and hence denser than ambient mantle. This would aid the long-term preservation of these mantle portions, and potentially points to their modern manifestation within seismically slow, deep mantle reservoirs with high 3He/4He ratios.

Community-wide correlations between species richness, abundance and population genomic diversity in a freshwater biodiversity hotspot.

Understanding patterns of diversity across macro (e.g. species-level) and micro (e.g. molecular-level) scales can shed light on community function and stability by elucidating the abiotic and biotic drivers of diversity within ecological communities. We examined the relationships among taxonomic and genetic metrics of diversity in freshwater mussels (Bivalvia: Unionidae), an ecologically important and species-rich group in the southeastern United States. Using quantitative community surveys and reduced-representation genome sequencing across 22 sites in seven rivers and two river basins, we surveyed 68 mussel species and sequenced 23 of these species to characterize intrapopulation genetic variation. We tested for the presence of species diversity-abundance correlations (i.e. the more-individuals hypothesis, MIH), species-genetic diversity correlations (SGDCs) and abundance-genetic diversity correlations (AGDCs) across all sites to evaluate relationships between different metrics of diversity. Sites with greater cumulative multispecies density (a standardized metric of abundance) had a greater number of species, consistent with the MIH hypothesis. Intrapopulation genetic diversity was strongly associated with the density of most species, indicating the presence of AGDCs. However, there was no consistent evidence for SGDCs. Although sites with greater overall densities of mussels had greater species richness, sites with higher genetic diversity did not always exhibit positive correlations with species richness, suggesting that there are spatial and evolutionary scales at which the processes influencing community-level diversity and intraspecific diversity differ. Our work reveals the importance of local abundance as indicator (and possibly a driver) of intrapopulation genetic diversity.

Microbial Composition of Freshwater Marsh Sediment Responds more Strongly to Microcosm Seawater Addition than Simulated Nitrate or Phosphate Eutrophication.

As sea level rise impacts coastal wetlands, saltmarsh will overtake coastal freshwater marsh in many areas, but changes in the sediment microbiome in response to saltwater intrusion are difficult to predict. Coastal freshwater marsh sediment was exposed to ambient, brackish, and saline conditions as well as to elevated nitrate and phosphate to model the combined stresses of saltwater intrusion and coastal eutrophication. Initially, sediment prokaryotic composition was similar to prior studies of freshwater marsh but diverged over time, reflecting the magnitude of increase in saltwater. There was no observed effect of nutrient amendment, potentially ranking seawater intrusion as a higher-importance compositional driver. Although the previously described loss of methanogenic populations and promotion of sulfate reducers in response to saltwater exposure was observed, taxonomic distribution was not similar to typical meso-polyhaline wetlands. Without colonization by marine taxa, such a community may be short-lived naturally, ultimately equilibrating with more common saltmarsh species. However, the recapitulation of salinity concentration by freshwater sediment microbial composition demonstrates the overwhelming nature of saltwater intrusion relative to other drivers like eutrophication.

Seasonal Patterns Contribute More Towards Phyllosphere Bacterial Community Structure than Short-Term Perturbations.

Phyllosphere microorganisms are sensitive to fluctuations in wind, temperature, solar radiation, and rain. However, recent explorations of patterns in phyllosphere communities across time often focus on seasonal shifts and leaf senescence without measuring the contribution of environmental drivers and leaf traits. Here, we focus on the effects of rain on the phyllosphere bacterial community of the wetland macrophyte broadleaf cattail (Typha latifolia) across an entire year, specifically targeting days before and 1, 3, and 5 days after rain events. To isolate the contribution of precipitation from other factors, we covered a subset of plants to shield them from rainfall. We used targeted Illumina sequencing of the V4 region of the bacterial 16S rRNA gene to characterize phyllosphere community composition. Rain events did not have a detectable effect on phyllosphere community richness or evenness regardless of whether the leaves were covered from rain or not, suggesting that foliar microbial communities are robust to such disturbances. While climatic and leaf-based variables effectively modeled seasonal trends in phyllosphere diversity and composition, they provided more limited explanatory value at shorter time scales. These findings underscore the dominance of long-term seasonal patterns related to climatic variation as the main factor influencing the phyllosphere community.

Functional importance and diversity of fungi during standing grass litter decomposition.

Although microbial participation in litter decomposition is widely known within terrestrial soils, the role and significance of microorganisms during the aerial standing litter phase of decomposition remains poorly investigated. We examined the fungi inhabiting standing leaf litter of Schizachyrium scoparium and Schizachyrium tenerum in a Longleaf Pine savanna ecosystem and estimated their contribution to litter decomposition. We identified fungal phylotypes associated with leaf litter and quantified leaf C mass loss, fungal biomass production, and microbial respiration during decomposition. These data were used to construct budgets estimating C flow into and through fungi. Significant losses in S. scoparium (55%) and S. tenerum (67%) leaf C mass were observed during standing decomposition along with concomitant increases in fungal biomass, which reached a maximum of 36 and 33 mgC/g detrital C, respectively. Cumulative fungal production during decomposition totaled 99 ± 6 mgC/g initial detrital C in S. scoparium and 73 ± 5 mgC/g initial detrital C in S. tenerum, indicating that 18 and 11% of the litter C was converted into fungal biomass, respectively. Corresponding estimates of cumulative fungal respiration totaled 106 ± 7 and 174 ± 11 mgC/g initial detrital C in S. scoparium and S. tenerum, respectively. Next generation sequencing identified several fungal phylotypes, with the majority of sequences belonging to the Ascomycota (Dothideomycetes) and Basidiomycota (Agaricomycetes). Fungal phylotypes were similar between litter species and changed over time, showing a successional pattern. These findings extend our understanding of fungal processes to standing litter in terrestrial ecosystems, and highlight the quantitative importance of fungi in C cycling processes.

Concentrations of select dissolved trace elements and anthropogenic organic compounds in the Mississippi River and major tributaries during the summer of 2012 and 2013.

The Mississippi River drainage basin includes the Illinois, Missouri, Ohio, Tennessee, and Arkansas rivers. These rivers drain areas with different physiography, population centers, and land use, with each contributing a different suites of metals and wastewater contaminants that can affect water quality. In July 2012, we determined 18 elements (Be, Rb, Sr, Cd, Cs, Ba, Tl, Pb, Mg, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn) and chlorophyll-a (Chl-a) in the five major tributaries and in the Upper Mississippi River. The following summer, we determined both trace elements and 25 trace organic compounds at 10 sites in a longitudinal study of the main stem of the Mississippi River from Grafton, Illinois to Natchez, Mississippi. We detected wastewater contaminants, including pharmaceuticals and endocrine disrupting compounds, throughout the river system, with the highest concentrations occurring near urban centers (St. Louis and Memphis). Concentrations were highest for atrazine (673 ng L-1), DEET (540 ng L-1), TCPP (231 ng L-1), and caffeine (202 ng L-1). The Illinois, Missouri, and Yazoo rivers, which drain areas with intense agriculture, had relatively high concentrations of Chl-a and atrazine. However, the Ohio River delivered higher loads of contaminants to the Mississippi River, including an estimated 177 kg day-1 of atrazine, due to higher flow volumes. Concentrations of heavy metals (Ni, V, Co, Cu, Cd, and Zn) were relatively high in the Illinois River and low in the Ohio River, although dissolved metal concentrations were below US EPA maximum contaminant levels for surface water. Multivariate analysis demonstrated that the rivers can be distinguished based on elemental and contaminant profiles.

Microbiome evolution along divergent branches of the vertebrate tree of life: what is known and unknown.

Vertebrates harbour microbes both internally and externally, and collectively, these microorganisms (the 'microbiome') contain genes that outnumber the host's genetic information 10-fold. The majority of the microorganisms associated with vertebrates are found within the gut, where they influence host physiology, immunity and development. The development of next-generation sequencing has led to a surge in effort to characterize the microbiomes of various vertebrate hosts, a necessary first step to determine the functional role these communities play in host evolution or ecology. This shift away from a culture-based microbiological approach, limited in taxonomic breadth, has resulted in the emergence of patterns suggesting a core vertebrate microbiome dominated by members of the bacterial phyla Bacteroidetes, Proteobacteria and Firmicutes. Still, there is a substantial variation in the methodology used to characterize the microbiome, from differences in sample type to issues of sampling captive or wild hosts, and the majority (>90%) of studies have characterized the microbiome of mammals, which represent just 8% of described vertebrate species. Here, we review the state of microbiome studies of nonmammalian vertebrates and provide a synthesis of emerging patterns in the microbiome of those organisms. We highlight the importance of collection methods, and the need for greater taxonomic sampling of natural rather than captive hosts, a shift in approach that is needed to draw ecologically and evolutionarily relevant inferences. Finally, we recommend future directions for vertebrate microbiome research, so that attempts can be made to determine the role that microbial communities play in vertebrate biology and evolution.

Biogeographic Patterns Between Bacterial Phyllosphere Communities of the Southern Magnolia (Magnolia grandiflora) in a Small Forest.

The phyllosphere presents a unique system of discrete and easily replicable surfaces colonized primarily by bacteria. However, the biogeography of bacteria in the phyllosphere is little understood, especially at small to intermediate scales. Bacterial communities on the leaves of 91 southern magnolia (Magnolia grandiflora) trees 1-452 m apart in a small forest plot were analyzed and fragments of the 16S ribosomal RNA (rRNA) gene sequenced using the Illumina platform. Assemblages were dominated by members of the Alphaproteobacteria, Bacteroidetes, and Acidobacteria. Patterns in community composition were measured by both relative abundance (theta) and presence-absence (Jaccard) dissimilarity metrics. Distance-based Moran's eigenvector map analyses of the distance-decay relationship found a significant, positive relationship between each dissimilarity metric and significant eigenfunctions derived from geographic distance between trees, indicating trees that were closer together had more similar bacterial phyllosphere communities. Indirect gradient analyses revealed that several environmental parameters (canopy cover, tree elevation, and the slope and aspect of the ground beneath trees) were significantly related to multivariate ordination scores based on relative bacterial sequence abundances; however, these relationships were not significant when looking at the incidence of bacterial taxa. This suggests that bacterial growth and abundance in the phyllosphere is shaped by different assembly mechanisms than bacterial presence or absence. More broadly, this study demonstrates that the distance-decay relationship applies to phyllosphere communities at local scales, and that environmental parameters as well as neutral forces may both influence spatial patterns in the phyllosphere.

Microbial Community Composition and Extracellular Enzyme Activities Associated with Juncus roemerianus and Spartina alterniflora Vegetated Sediments in Louisiana Saltmarshes.

Saltmarshes are typically dominated by perennial grasses with large underground rhizome systems that can change local sediment conditions and be important in shaping the sediment microbial community. Factors such as salinity that control plant zonation in saltmarshes are also likely to influence the microbial community, but little is known as to whether microbial communities share distribution patterns with plants in these systems. To determine the extent to which microbial assemblages are influenced by saltmarsh plant communities, as well as to examine patterns in microbial community structure at local and regional scales, we sampled sediments at three saltmarshes in Louisiana, USA. All three systems exhibit a patchy distribution of Juncus roemerianus stands within a Spartina alterniflora marsh. Sediment samples were collected from the interior of several J. roemerianus stands as well as from the S. alterniflora matrix. Samples were assayed for extracellular enzyme activity and DNA extracted to determine microbial community composition. Denaturing gradient gel electrophoresis of rRNA gene fragments was used to determine regional patterns in bacterial, archaeal, and fungal assemblages, while Illumina sequencing was used to examine local, vegetation-driven, patterns in community structure at one site. Both enzyme activity and microbial community structure were primarily influenced by regional site. Within individual saltmarshes, bacterial and archaeal communities differed between J. roemerianus and S. alterniflora vegetated sediments, while fungal communities did not. These results highlight the importance of the plant community in shaping the sediment microbial community in saltmarshes but also demonstrate that regional scale factors are at least as important.

Activities and Prevalence of Proteobacteria Members Colonizing Echinacea purpurea Fully Account for Macrophage Activation Exhibited by Extracts of This Botanical.

Evidence supports the theory that bacterial communities colonizing Echinacea purpurea contribute to the innate immune enhancing activity of this botanical. Previously, we reported that only about half of the variation in in vitro monocyte stimulating activity exhibited by E. purpurea extracts could be accounted for by total bacterial load within the plant material. In the current study, we test the hypothesis that the type of bacteria, in addition to bacterial load, is necessary to fully account for extract activity. Bacterial community composition within commercial and freshly harvested (wild and cultivated) E. purpurea aerial samples was determined using high-throughput 16S rRNA gene pyrosequencing. Bacterial isolates representing 38 different taxa identified to be present within E. purpurea were acquired, and the activity exhibited by the extracts of these isolates varied by over 8000-fold. Members of the Proteobacteria exhibited the highest potency for in vitro macrophage activation and were the most predominant taxa. Furthermore, the mean activity exhibited by the Echinacea extracts could be solely accounted for by the activities and prevalence of Proteobacteria members comprising the plant-associated bacterial community. The efficacy of E. purpurea material for use against respiratory infections may be determined by the Proteobacterial community composition of this plant, since ingestion of bacteria (probiotics) is reported to have a protective effect against this health condition.

Free-Living and Particle-Associated Bacterioplankton in Large Rivers of the Mississippi River Basin Demonstrate Biogeographic Patterns.

The different drainage basins of large rivers such as the Mississippi River represent interesting systems in which to study patterns in freshwater microbial biogeography. Spatial variability in bacterioplankton communities in six major rivers (the Upper Mississippi, Missouri, Illinois, Ohio, Tennessee, and Arkansas) of the Mississippi River Basin was characterized using Ion Torrent 16S rRNA amplicon sequencing. When all systems were combined, particle-associated (>3 µm) bacterial assemblages were found to be different from free-living bacterioplankton in terms of overall community structure, partly because of differences in the proportional abundance of sequences affiliated with major bacterial lineages (Alphaproteobacteria, Cyanobacteria, and Planctomycetes). Both particle-associated and free-living communities ordinated by river system, a pattern that was apparent even after rare sequences or those affiliated with Cyanobacteria were removed from the analyses. Ordination of samples by river system correlated with environmental characteristics of each river, such as nutrient status and turbidity. Communities in the Upper Mississippi and the Missouri and in the Ohio and the Tennessee, pairs of rivers that join each other, contained similar taxa in terms of presence-absence data but differed in the proportional abundance of major lineages. The most common sequence types detected in particle-associated communities were picocyanobacteria in the Synechococcus/Prochlorococcus/Cyanobium (Syn/Pro) clade, while free-living communities also contained a high proportion of LD12 (SAR11/Pelagibacter)-like Alphaproteobacteria. This research shows that while different tributaries of large river systems such as the Mississippi River harbor distinct bacterioplankton communities, there is also microhabitat variation such as that between free-living and particle-associated assemblages.

Culture dependent and independent analysis of bacterial communities associated with commercial salad leaf vegetables.

BACKGROUND: Plants harbor a diverse bacterial community, both as epiphytes on the plant surface and as endophytes within plant tissue. While some plant-associated bacteria act as plant pathogens or promote plant growth, others may be human pathogens. The aim of the current study was to determine the bacterial community composition of organic and conventionally grown leafy salad vegetables at the point of consumption using both culture-dependent and culture-independent methods. RESULTS: Total culturable bacteria on salad vegetables ranged from 8.0 × 10(3) to 5.5 × 10(8) CFU g(-1). The number of culturable endophytic bacteria from surface sterilized plants was significantly lower, ranging from 2.2 × 10(3) to 5.8 × 10(5) CFU g(-1). Cultured isolates belonged to six major bacterial phyla, and included representatives of Pseudomonas, Pantoea, Chryseobacterium, and Flavobacterium. Eleven different phyla and subphyla were identified by culture-independent pyrosequencing, with Gammaproteobacteria, Betaproteobacteria, and Bacteroidetes being the most dominant lineages. Other bacterial lineages identified (e.g. Firmicutes, Alphaproteobacteria, Acidobacteria, and Actinobacteria) typically represented less than 1% of sequences obtained. At the genus level, sequences classified as Pseudomonas were identified in all samples and this was often the most prevalent genus. Ralstonia sequences made up a greater portion of the community in surface sterilized than non-surface sterilized samples, indicating that it was largely endophytic, while Acinetobacter sequences appeared to be primarily associated with the leaf surface. Analysis of molecular variance indicated there were no significant differences in bacterial community composition between organic versus conventionally grown, or surface-sterilized versus non-sterilized leaf vegetables. While culture-independent pyrosequencing identified significantly more bacterial taxa, the dominant taxa from pyrosequence data were also detected by traditional culture-dependent methods. CONCLUSIONS: The use of pyrosequencing allowed for the identification of low abundance bacteria in leaf salad vegetables not detected by culture-dependent methods. The presence of a range of bacterial populations as endophytes presents an interesting phenomenon as these microorganisms cannot be removed by washing and are thus ingested during salad consumption.

Total bacterial load within Echinacea purpurea, determined using a new PCR-based quantification method, is correlated with LPS levels and in vitro macrophage activity.

Our previous studies indicate that the majority of in vitro monocyte/macrophage activation exhibited by extracts of Echinacea depends on bacterial components. In the present study, total bacterial load was determined within E. purpurea samples and ranged from 6.4 × 10(6) to 3.3 × 10(8) bacteria/g of dry plant material. To estimate total bacterial load, we developed a PCR-based quantification method that circumvents the problems associated with nonviable/nonculturable cells (which precludes using plate counts) or the coamplification of mitochondrial or chloroplast DNA with the use of universal bacterial primers (which precludes the use of qPCR). Differences in total bacterial load within Echinacea samples were strongly correlated with the activity (NF-κB activation in THP-1 cells) and content of bacterial lipopolysaccharides within extracts of this plant material. These results add to the growing body of evidence that bacteria within Echinacea are the main source of components responsible for enhancing innate immune function.

Determining potential for microbial atrazine degradation in agricultural drainage ditches.

Passage of agricultural runoff through vegetated drainage ditches has been shown to reduce the amount of pesticides, such as atrazine, exiting out of agricultural watersheds. Previous studies have found that microbial communities in soil from fields treated with atrazine display enhanced rates of atrazine degradation. However, no studies have examined the potential for atrazine degradation in ditches used to drain these lands. The purpose of the current study was to determine the potential of the drainage ditch soil microbial community for atrazine degradation. Soil samples were collected from fields and adjacent drainage ditches and from nonagricultural land with no previous exposure to atrazine. Polymerase chain reaction analysis indicated widespread presence of atrazine degradation genes in fields and ditches. Potential for degradation was determined by following the decrease of atrazine in spiked soil samples over a 28-d incubation period. Greater than 95% of atrazine was degraded in field and ditch soils, whereas only 68.5 ± 1.3% was degraded in the nonagricultural control. Comparison with autoclaved soil samples indicated the primary mechanism of atrazine degradation in agricultural soils was microbially mediated, whereas its breakdown in nonagricultural soil appeared to be the byproduct of abiotic processes. Therefore, microbial communities in drainage ditch sediments have the potential to play a role in atrazine removal from agricultural runoff by breaking down atrazine deposited in sediments and limiting the amount of this herbicide carried into downstream ecosystems.

Rapid transition from primary to secondary crust building on the Moon explained by mantle overturn.

Geochronology indicates a rapid transition (tens of Myrs) from primary to secondary crust building on the Moon. The processes responsible for initiating secondary magmatism, however, remain in debate. Here we test the hypothesis that the earliest secondary crust (Mg-suite) formed as a direct consequence of density-driven mantle overturn, and advance 3D mantle convection models to quantify the resulting extent of lower mantle melting. Our modeling demonstrates that overturn of thin ilmenite-bearing cumulates ≤ 100 km triggers a rapid and short-lived episode of lower mantle melting which explains the key volume, geochronological, and spatial characteristics of early secondary crust building without contributions from other energy sources, namely KREEP (potassium, rare earth elements, phosphorus, radiogenic U, Th). Observations of globally distributed Mg-suite eliminate degree-1 overturn scenarios. We propose that gravitational instabilities in magma ocean cumulate piles are major driving forces for the onset of mantle convection and secondary crust building on differentiated bodies.

Characterizing the Gut Microbial Communities of Native and Invasive Freshwater Bivalves after Long-Term Sample Preservation.

Freshwater mussels are important indicators of the overall health of their environment but have suffered declines that have been attributed to factors such as habitat degradation, a loss of fish hosts, climate change, and excessive nutrient inputs. The loss of mussel biodiversity can negatively impact freshwater ecosystems such that understanding the mussel's gut microbiome has been identified as a priority topic for developing conservation strategies. In this study, we determine whether ethanol-stored specimens of freshwater mussels can yield representative information about their gut microbiomes such that changes in the microbiome through time could potentially be determined from museum mussel collections. A short-term preservation experiment using the invasive clam Corbicula fluminea was used to validate the use of ethanol as a method for storing the bivalve microbiome, and the gut microbiomes of nine native mussel species that had been preserved in ethanol for between 2 and 9 years were assessed. We show that ethanol preservation is a valid storage method for bivalve specimens in terms of maintaining an effective sequencing depth and the richness of their gut bacterial assemblages and provide further insight into the gut microbiomes of the invasive clam C. fluminea and nine species of native mussels. From this, we identify a "core" genus of bacteria (Romboutsia) that is potentially common to all freshwater bivalve species studied. These findings support the potential use of ethanol-preserved museum specimens to examine patterns in the gut microbiomes of freshwater mussels over long periods.

Evaluating Methods of Preserving Aquatic Invertebrates for Microbiome Analysis.

Research on the microbiomes of animals has increased substantially within the past decades. More recently, microbial analyses of aquatic invertebrates have become of increased interest. The storage method used while collecting aquatic invertebrates has not been standardized throughout the scientific community, and the effects of common storage methods on the microbial composition of the organism is unknown. Using crayfish and dragonfly nymphs collected from a natural pond and crayfish maintained in an aquarium, the effects of two common storage methods, preserving in 95% ethanol and freezing at -20 °C, on the invertebrate bacterial microbiome was evaluated. We found that the bacterial community was conserved for two sample types (gut and exoskeleton) of field-collected crayfish stored either in ethanol or frozen, as was the gut microbiome of aquarium crayfish. However, there were significant differences between the bacterial communities found on the exoskeleton of aquarium crayfish stored in ethanol compared to those that were frozen. Dragonfly nymphs showed significant differences in gut microbial composition between species, but the microbiome was conserved between storage methods. These results demonstrate that preserving field-collected specimens of aquatic invertebrates in 95% ethanol is likely to be a simple and effective sample preservation method for subsequent gut microbiome analysis but is less reliable for the external microbiome.

Microbial Communities in Saltpan Sediments Show Tolerance to Mars Analog Conditions, but Susceptibility to Chloride and Perchlorate Toxicity.

Brines at or near the surface of present-day Mars are a potential explanation for seasonally recurring dark streaks on the walls of craters, termed recurring slope lineae (RSL). Deliquescence and freezing point depression are possible drivers of brine stability, attributable to the high salinity observed in martian regolith including chlorides and perchlorates. Investigation of life, which may inhabit RSL, and the cellular mechanisms necessary for survival, must consider the tolerance of highly variable hydration, freeze-thaw cycles, and high osmolarity in addition to the anaerobic, oligotrophic, and irradiated environment. We propose the saltpan, an ephemeral, hypersaline wetland as an analogue for putative RSL hydrology. Saltpan sediment archaeal and bacterial communities showed tolerance of the Mars-analogous atmosphere, hydration, minerology, salinity, and temperature. Although active growth and a shift to well-adapted taxa were observed, susceptibility to low-concentration chloride and perchlorate addition suggested that such a composition was insufficient for beneficial water retention relative to added salt stress.

Ranking the biases: The choice of OTUs vs. ASVs in 16S rRNA amplicon data analysis has stronger effects on diversity measures than rarefaction and OTU identity threshold.

Advances in the analysis of amplicon sequence datasets have introduced a methodological shift in how research teams investigate microbial biodiversity, away from sequence identity-based clustering (producing Operational Taxonomic Units, OTUs) to denoising methods (producing amplicon sequence variants, ASVs). While denoising methods have several inherent properties that make them desirable compared to clustering-based methods, questions remain as to the influence that these pipelines have on the ecological patterns being assessed, especially when compared to other methodological choices made when processing data (e.g. rarefaction) and computing diversity indices. We compared the respective influences of two widely used methods, namely DADA2 (a denoising method) vs. Mothur (a clustering method) on 16S rRNA gene amplicon datasets (hypervariable region v4), and compared such effects to the rarefaction of the community table and OTU identity threshold (97% vs. 99%) on the ecological signals detected. We used a dataset comprising freshwater invertebrate (three Unionidae species) gut and environmental (sediment, seston) communities sampled in six rivers in the southeastern USA. We ranked the respective effects of each methodological choice on alpha and beta diversity, and taxonomic composition. The choice of the pipeline significantly influenced alpha and beta diversities and changed the ecological signal detected, especially on presence/absence indices such as the richness index and unweighted Unifrac. Interestingly, the discrepancy between OTU and ASV-based diversity metrics could be attenuated by the use of rarefaction. The identification of major classes and genera also revealed significant discrepancies across pipelines. Compared to the pipeline's effect, OTU threshold and rarefaction had a minimal impact on all measurements.

Environment and Co-occurring Native Mussel Species, but Not Host Genetics, Impact the Microbiome of a Freshwater Invasive Species (Corbicula fluminea).

The Asian clam Corbicula fluminea (Family: Cyneridae) has aggressively invaded freshwater habitats worldwide, resulting in dramatic ecological changes and declines of native bivalves such as freshwater mussels (Family: Unionidae), one of the most imperiled faunal groups. Despite increases in our knowledge of invasive C. fluminea biology, little is known of how intrinsic and extrinsic factors, including co-occurring native species, influence its microbiome. We investigated the gut bacterial microbiome across genetically differentiated populations of C. fluminea in the Tennessee and Mobile River Basins in the Southeastern United States and compared them to those of six co-occurring species of native freshwater mussels. The gut microbiome of C. fluminea was diverse, differed with environmental conditions and varied spatially among rivers, but was unrelated to host genetic variation. Microbial source tracking suggested that the gut microbiome of C. fluminea may be influenced by the presence of co-occurring native mussels. Inferred functions from 16S rRNA gene data using PICRUST2 predicted a high prevalence and diversity of degradation functions in the C. fluminea microbiome, especially the degradation of carbohydrates and aromatic compounds. Such modularity and functional diversity of the microbiome of C. fluminea may be an asset, allowing to acclimate to an extensive range of nutritional sources in invaded habitats, which could play a vital role in its invasive success.