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.

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.

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.

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.

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.

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.

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.

Tracing the origin of the crayfish plague pathogen, Aphanomyces astaci, to the Southeastern United States.

The oomycete Aphanomyces astaci is an emerging infectious pathogen affecting freshwater crayfish worldwide and is responsible for one of the most severe wildlife pandemics ever reported. The pathogen has caused mass mortalities of freshwater crayfish species in Europe and Asia, and threatens other susceptible species in Madagascar, Oceania and South America. The pathogen naturally coexists with some North American crayfish species that are its chronic carriers. Presumptions that A. astaci originated in North America are based on disease outbreaks that followed translocations of North American crayfish and on the identification of the pathogen mainly in Europe. We studied A. astaci in the southeastern US, a center of freshwater crayfish diversity. In order to decipher the origin of the pathogen, we investigated (1) the distribution and haplotype diversity of A. astaci, and (2) whether there are crayfish species-specificities and/or geographical restrictions for A. astaci haplotypes. A total of 132 individuals, corresponding to 19 crayfish species and one shrimp species from 23 locations, tested positive for A. astaci. Mitochondrial rnnS and rnnL sequences indicated that A. astaci from the southeastern US exhibited the highest genetic diversity so far described for the pathogen (eight haplotypes, six of which we newly describe). Our findings that A. astaci is widely distributed and genetically diverse in the region supports the hypothesis that the pathogen originated in the southeastern US. In contrast to previous assumptions, however, the pathogen exhibited no clear species-specificity or geographical patterns.

Gut Microbiomes of Freshwater Mussels (Unionidae) Are Taxonomically and Phylogenetically Variable across Years but Remain Functionally Stable.

Freshwater mussels perform essential ecosystem functions, yet we have no information on how their microbiomes fluctuate over time. In this study, we examined temporal variation in the microbiome of six mussel species (Lampsilis ornata, Obovaria unicolor, Elliptio arca, Fusconaia cerina, Cyclonaias asperata, and Tritogonia verrucosa) sampled from the same river in 2016 and 2019. We examined the taxonomic, phylogenetic, and inferred functional (from 16S rRNA sequences) facets of their microbiome diversity. Significant differences between the two years were identified in five of the six species sampled. However, not all species that exhibited a temporally variable microbiome were functionally distinct across years, indicating functional redundancy within the mussel gut microbiome. Inferred biosynthesis pathways showed temporal variation in pathways involved in degradation, while pathways involved in cellular metabolism were stable. There was no evidence for phylosymbiosis across any facet of microbiome biodiversity. These results indicate that temporal variation is an important factor in the assembly of the gut microbiomes of freshwater mussels and provides further support that the mussel gut microbiome is involved in host development and activity.

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.

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.

Gut microbiomes of sympatric Amazonian wood-eating catfishes (Loricariidae) reflect host identity and little role in wood digestion.

Neotropical wood-eating catfishes (family Loricariidae) can occur in diverse assemblages with multiple genera and species feeding on the same woody detritus. As such, they present an intriguing system in which to examine the influence of host species identity on the vertebrate gut microbiome as well as to determine the potential role of gut bacteria in wood digestion. We characterized the gut microbiome of two co-occurring catfish genera and four species: Panaqolus albomaculatus, Panaqolus gnomus, Panaqolus nocturnus, and Panaque bathyphilus, as well as that of submerged wood on which they feed. The gut bacterial community did not significantly vary across three gut regions (proximal, mid, distal) for any catfish species, although interspecific variation in the gut microbiome was significant, with magnitude of interspecific difference generally reflecting host phylogenetic proximity. Further, the gut microbiome of each species was significantly different to that present on the submerged wood. Inferring the genomic potential of the gut microbiome revealed that the majority of wood digesting pathways were at best equivalent to and more often depleted or nonexistent within the catfish gut compared to the submerged wood, suggesting a minimal role for the gut microbiome in wood digestion. Rather, these fishes are more likely reliant on fiber degradation performed by microbes in the environment, with their gut microbiome determined more by host identity and phylogenetic history.

Microbiomes of Caribbean Octocorals Vary Over Time but Are Resistant to Environmental Change.

The bacterial microbiome is an essential component of many corals, although knowledge of the microbiomes in scleractinian corals far exceeds that for octocorals. This study characterized the bacterial communities present in shallow water Caribbean gorgonian octocorals over time and space, in addition to determining the bacterial assemblages in gorgonians exposed to environmental perturbations. We found that seven shallow water Caribbean gorgonian species maintained distinct microbiomes and predominantly harbored two bacterial genera, Mycoplasma and Endozoicomonas. Representatives of these taxa accounted for over 70% of the sequences recovered, made up the three most common operational taxonomic units (OTUs), and were present in most of the gorgonian species. Gorgonian species sampled in different seasons and/or in different years, exhibited significant shifts in the abundances of these bacterial OTUs, though there were few changes to overall bacterial diversity, or to the specific OTUs present. These shifts had minimal impact on the relative abundance of inferred functional proteins within the gorgonian corals. Sequences identified as Escherichia were ubiquitous in gorgonian colonies sampled from a lagoon but not in colonies sampled from a back reef. Exposure to increased temperature and/or ultraviolet radiation (UVR) or nutrient enrichment led to few significant changes in the gorgonian coral microbiomes. While there were some shifts in the abundance of the prevalent bacteria, more commonly observed was "microbial switching" between different OTUs identified within the same bacterial genus. The relative stability of gorgonian coral bacterial microbiome may potentially explain some of the resistance and resilience of Caribbean gorgonian corals against changing environmental conditions.

Timescales of variation in diversity and production of bacterioplankton assemblages in the Lower Mississippi River.

Rivers are characterized by rapid and continuous one-way directional fluxes of flowing, aqueous habitat, chemicals, suspended particles, and resident plankton. Therefore, at any particular location in such systems there is the potential for continuous, and possibly abrupt, changes in diversity and metabolic activities of suspended biota. As microorganisms are the principal catalysts of organic matter degradation and nutrient cycling in rivers, examination of their assemblage dynamics is fundamental to understanding system-level biogeochemical patterns and processes. However, there is little known of the dynamics of microbial assemblage composition or production of large rivers along a time interval gradient. We quantified variation in alpha and beta diversity and production of particle-associated and free-living bacterioplankton assemblages collected at a single site on the Lower Mississippi River (LMR), the final segment of the largest river system in North America. Samples were collected at timescales ranging from days to weeks to months up to a year. For both alpha and beta diversity, there were similar patterns of temporal variation in particle-associated and free-living assemblages. Alpha diversity, while always higher on particles, varied as much at a daily as at a monthly timescale. Beta diversity, in contrast, gradually increased with time interval of sampling, peaking between samples collected 180 days apart, before gradually declining between samples collected up to one year apart. The primary environmental driver of the temporal pattern in beta diversity was temperature, followed by dissolved nitrogen and chlorophyll a concentrations. Particle-associated bacterial production corresponded strongly to temperature, while free-living production was much lower and constant over time. We conclude that particle-associated and free-living bacterioplankton assemblages of the LMR vary in richness, composition, and production at distinct timescales in response to differing sets of environmental factors. This is the first temporal longitudinal study of microbial assemblage structure and dynamics in the LMR.

The Saltpan Microbiome is Structured by Sediment Depth and Minimally Influenced by Variable Hydration.

Saltpans are a class of ephemeral wetland characterized by alternating periods of inundation, rising salinity, and desiccation. We obtained soil cores from a saltpan on the Mississippi Gulf coast in both the inundated and desiccated state. The microbiomes of surface and 30 cm deep sediment were determined using Illumina sequencing of the V4 region of the 16S rRNA gene. Bacterial and archaeal community composition differed significantly between sediment depths but did not differ between inundated and desiccated states. Well-represented taxa included marine microorganisms as well as multiple halophiles, both observed in greater proportions in surface sediment. Functional inference of metagenomic data showed that saltpan sediments in the inundated state had greater potential for microbial activity and that several energetic and degradation pathways were more prevalent in saltpan sediment than in nearby tidal marsh sediment. Microbial communities within saltpan sediments differed in composition from those in adjacent freshwater and brackish marshes. These findings indicate that the bacterial and archaeal microbiomes of saltpans are highly stratified by sediment depth and are only minimally influenced by changes in hydration. The surface sediment community is likely isolated from the shallow subsurface community by compaction, with the microbial community dominated by marine and terrestrial halophiles.

Effects of Life Stage, Site, and Species on the Dragonfly Gut Microbiome.

Insects that undergo metamorphosis from juveniles to adults provide an intriguing opportunity to examine the effects of life stage, species, and the environment on their gut microbiome. In this study, we surveyed the gut microbiomes of 13 species of dragonfly collected from five different locations subject to different levels of human impact. Juveniles were collected as nymphs from aquatic habitats while airborne adults were caught at the same locations. The gut microbiome was characterized by next generation sequencing of the bacterial 16S rRNA gene. Life stage was an important factor, with the gut microbiomes of dragonfly nymphs differing from those of adult dragonflies. Gut microbiomes of nymphs were influenced by sample site and, to a lesser extent, host species. Neither sample location nor host species had a strong effect on the gut microbiome of dragonfly adults. Regardless of life stage, gut microbiomes were dominated by members of the Proteobacteria, with members of the Bacteroidetes (especially in adults), Firmicutes, and Acidobacteria (especially in nymphs) also being proportionally abundant. These results demonstrate that different life stages of metamorphosing insects can harbor very different gut microbiomes and differ in how this microbiome is influenced by the surrounding environment.

Macrophage activation by edible mushrooms is due to the collaborative interaction of toll-like receptor agonists and dectin-1b activating beta glucans derived from colonizing microorganisms.

Research supports the theory that the microbiome of plants and mushrooms produce potent activators of pathogen recognition receptors which are principal contributors to the stimulation of macrophages. We have previously reported that the in vitro macrophage stimulatory activity of water-soluble extracts from 13 different types of edible mushrooms is predominantly due to bacterial components originating from the naturally occurring bacterial communities within these materials. The purpose of the current study was to further investigate the bacterial-dependent activity of the water-soluble extracts and assess whether these 13 types of mushrooms contain water-insoluble beta glucans that activate the dectin-1b signaling pathway. Activity of the water-soluble extracts was predominantly due to Toll-like receptor 2 (TLR2) and TLR4 agonists. For dectin-1b-dependent activity (indicative of water-insoluble beta glucans), culinary mushrooms (Agaricus bisporus varieties) were essentially inactive, whereas most of the medicinal mushrooms (Lentinula edodes, Grifola frondosa, Hypsizygus marmoreus varieties, Flammulina velutipes) exhibited potent activation. A. bisporus samples with no detectable dectin-1b-dependent activity had yeast colony forming units that were 687 times lower than L. edodes exhibiting high activity, indicating that the active insoluble beta glucans are derived from colonizing yeast. In addition, co-stimulation of macrophages with the TLR agonists and insoluble beta glucan was found to result in a synergistic enhancement of in vitro cytokine production. Taken together, these findings indicate that the in vitro macrophage activating potential of edible mushrooms is due to the collaborative interaction of water-soluble TLR agonists (derived from colonizing bacteria) and water-insoluble beta glucans (derived from colonizing yeast).

The gut microbiome of freshwater Unionidae mussels is determined by host species and is selectively retained from filtered seston.

Freshwater mussels are a species-rich group of aquatic invertebrates that are among the most endangered groups of fauna worldwide. As filter-feeders that are constantly exposed to new microbial inoculants, mussels represent an ideal system to investigate the effects of species or the environment on gut microbiome composition. In this study, we examined if host species or site exerts a greater influence on microbiome composition. Individuals of four co-occurring freshwater mussel species, Cyclonaias asperata, Fusconaia cerina, Lampsilis ornata, and Obovaria unicolor were collected from six sites along a 50 km stretch of the Sipsey River in Alabama, USA. High throughput 16S rRNA gene sequencing revealed that mussel gut bacterial microbiota were distinct from bacteria on seston suspended in the water column, and that the composition of the gut microbiota was influenced by both host species and site. Despite species and environmental variation, the most frequently detected sequences within the mussel microbiota were identified as members of the Clostridiales. Sequences identified as the nitrogen-fixing taxon Methylocystis sp. were also abundant in all mussel species, and sequences of both bacterial taxa were more abundant in mussels than in water. Site physicochemical conditions explained almost 45% of variation in seston bacterial communities but less than 8% of variation in the mussel bacterial microbiome. Together, these findings suggest selective retention of bacterial taxa by the freshwater mussel host, and that both species and the environment are important in determining mussel gut microbiome composition.

Canopy position is a stronger determinant of bacterial community composition and diversity than environmental disturbance in the phyllosphere.

The effect of rain on the phyllosphere community has not been extensively explored, especially in the context of spatial variation on the impact of rain throughout the tree canopy. We characterized the response of the phyllosphere bacterial community removed from leaf surfaces of the Southern Magnolia (Magnolia grandiflora) to rain across different spatial locations of the canopy. We hypothesized that: (i) rain would lead to an initial decrease in phyllosphere bacterial diversity, followed by an increase in diversity on subsequent days, but that this effect would be minimized in the lower and interior portion of the canopy, and that (ii) community beta dispersion of phyllosphere microorganisms would be lower following rain, and similarly contingent on canopy position. We used targeted next-generation sequencing of the V4 region of the bacterial 16S rRNA gene to characterize bacterial composition. We found higher bacterial richness in interior canopy and distinct composition across canopy positions. Further, the effect of rain on beta dispersion was contingent on canopy position: rain lowered dispersion in the upper canopy but increased it in the lower and interior canopy. Our results demonstrate that canopy structure should be considered when looking at the impact of rain on the collective phyllosphere community.