Homogeneous Environmental Selection Structures the Bacterial Communities of Benthic Biofilms in Proglacial Floodplain Streams.

In proglacial floodplains, glacier recession promotes biogeochemical and ecological gradients across relatively small spatial scales. The resulting environmental heterogeneity induces remarkable microbial biodiversity among proglacial stream biofilms. Yet the relative importance of environmental constraints in forming biofilm communities remains largely unknown. Extreme environmental conditions in proglacial streams may lead to the homogenizing selection of biofilm-forming microorganisms. However, environmental differences between proglacial streams may impose different selective forces, resulting in nested, spatially structured assembly processes. Here, we investigated bacterial community assembly processes by unraveling ecologically successful phylogenetic clades in two stream types (glacier-fed mainstems and non-glacier-fed tributaries) draining three proglacial floodplains in the Swiss Alps. Clades with low phylogenetic turnover rates were present in all stream types, including Gammaproteobacteria and Alphaproteobacteria, while the other clades were specific to one stream type. These clades constituted up to 34.8% and 31.1% of the community diversity and up to 61.3% and 50.9% of the relative abundances in mainstems and tributaries, respectively, highlighting their importance and success in these communities. Furthermore, the proportion of bacteria under homogeneous selection was inversely related to the abundance of photoautotrophs, and these clades may therefore decrease in abundance with the future "greening" of proglacial habitats. Finally, we found little effect of physical distance from the glacier on clades under selection in glacier-fed streams, probably due to the high hydrological connectivity of our study reaches. Overall, these findings shed new light on the mechanisms of microbial biofilm assembly in proglacial streams and help us to predict their future in a rapidly changing environment. IMPORTANCE Streams draining proglacial floodplains harbor benthic biofilms comprised of diverse microbial communities. These high-mountain ecosystems are rapidly changing with climate warming, and it is therefore critical to better understand the mechanisms underlying the assembly of their microbial communities. We found that homogeneous selection dominates the structuring of bacterial communities in benthic biofilms in both glacier-fed mainstems and nonglacier tributary streams within three proglacial floodplains in the Swiss Alps. However, differences between glacier-fed and tributary ecosystems may impose differential selective forces. Here, we uncovered nested, spatially structured assembly processes for proglacial floodplain communities. Our analyses additionally provided insights into linkages between aquatic photoautotrophs and the bacterial taxa under homogeneous selection, potentially by providing a labile source of carbon in these otherwise carbon-deprived systems. In the future, we expect a shift in the bacterial communities under homogeneous selection in glacier-fed streams as primary production becomes more important and streams become "greener".

Benthic Biofilms in Glacier-Fed Streams from Scandinavia to the Himalayas Host Distinct Bacterial Communities Compared with the Streamwater.

Microbial life in glacier-fed streams (GFSs) is dominated by benthic biofilms which fulfill critical ecosystem processes. However, it remains unclear how the bacterial communities of these biofilms assemble in stream ecosystems characterized by rapid turnover of benthic habitats and high suspended sediment loads. Using16S rRNA gene amplicon sequence data collected from 54 GFSs across the Himalayas, European Alps, and Scandinavian Mountains, we found that benthic biofilms harbor bacterial communities that are distinct from the bacterial assemblages suspended in the streamwater. Our data showed a decrease in species richness in the benthic biofilms compared to the bacterial cells putatively free-living in the water. The benthic biofilms also differed from the suspended water fractions in terms of community composition. Differential abundance analyses highlighted bacterial families that were specific to the benthic biofilms and the suspended assemblages. Notably, source-sink models suggested that the benthic biofilm communities are not simply a subset of the suspended assemblages. Rather, we found evidence that deterministic processes (e.g., species sorting) shape the benthic biofilm communities. This is unexpected given the high vertical mixing of water and contained bacterial cells in GFSs and further highlights the benthic biofilm mode of life as one that is determined through niche-related processes. Our findings therefore reveal a "native" benthic biofilm community in an ecosystem that is currently threatened by climate-induced glacier shrinkage. IMPORTANCE Benthic biofilms represent the dominant form of life in glacier-fed streams. However, it remains unclear how bacterial communities within these biofilms assemble. Our findings from glacier-fed streams from three major mountain ranges across the Himalayas, the European Alps and the Scandinavian Mountains reveal a bacterial community associated with benthic biofilms that is distinct from the assemblage in the overlying streamwater. Our analyses suggest that selection is the underlying process to this differentiation. This is unexpected given that bacterial cells that are freely living or attached to the abundant sediment particles suspended in the water continuously mix with the benthic biofilms. The latter colonize loose sediments that are subject to high turnover owing to the forces of the water flow. Our research unravels the existence of a microbiome specific to benthic biofilms in glacier-fed streams, now under major threats due to global warming.

Glacier shrinkage will accelerate downstream decomposition of organic matter and alters microbiome structure and function.

The shrinking of glaciers is among the most iconic consequences of climate change. Despite this, the downstream consequences for ecosystem processes and related microbiome structure and function remain poorly understood. Here, using a space-for-time substitution approach across 101 glacier-fed streams (GFSs) from six major regions worldwide, we investigated how glacier shrinkage is likely to impact the organic matter (OM) decomposition rates of benthic biofilms. To do this, we measured the activities of five common extracellular enzymes and estimated decomposition rates by using enzyme allocation equations based on stoichiometry. We found decomposition rates to average 0.0129 (% d-1 ), and that decreases in glacier influence (estimated by percent glacier catchment coverage, turbidity, and a glacier index) accelerates decomposition rates. To explore mechanisms behind these relationships, we further compared decomposition rates with biofilm and stream water characteristics. We found that chlorophyll-a, temperature, and stream water N:P together explained 61% of the variability in decomposition. Algal biomass, which is also increasing with glacier shrinkage, showed a particularly strong relationship with decomposition, likely indicating their importance in contributing labile organic compounds to these carbon-poor habitats. We also found high relative abundances of chytrid fungi in GFS sediments, which putatively parasitize these algae, promoting decomposition through a fungal shunt. Exploring the biofilm microbiome, we then sought to identify bacterial phylogenetic clades significantly associated with decomposition, and found numerous positively (e.g., Saprospiraceae) and negatively (e.g., Nitrospira) related clades. Lastly, using metagenomics, we found evidence of different bacterial classes possessing different proportions of EEA-encoding genes, potentially informing some of the microbial associations with decomposition rates. Our results, therefore, present new mechanistic insights into OM decomposition in GFSs by demonstrating that an algal-based "green food web" is likely to increase in importance in the future and will promote important biogeochemical shifts in these streams as glaciers vanish.

Spatial patterns of benthic biofilm diversity among streams draining proglacial floodplains.

Glacier shrinkage opens new proglacial terrain with pronounced environmental gradients along longitudinal and lateral chronosequences. Despite the environmental harshness of the streams that drain glacier forelands, their benthic biofilms can harbor astonishing biodiversity spanning all domains of life. Here, we studied the spatial dynamics of prokaryotic and eukaryotic photoautotroph diversity within braided glacier-fed streams and tributaries draining lateral terraces predominantly fed by groundwater and snowmelt across three proglacial floodplains in the Swiss Alps. Along the lateral chronosequence, we found that benthic biofilms in tributaries develop higher biomass than those in glacier-fed streams, and that their respective diversity and community composition differed markedly. We also found spatial turnover of bacterial communities in the glacier-fed streams along the longitudinal chronosequence. These patterns along the two chronosequences seem unexpected given the close spatial proximity and connectivity of the various streams, suggesting environmental filtering as an underlying mechanism. Furthermore, our results suggest that photoautotrophic communities shape bacterial communities across the various streams, which is understandable given that algae are the major source of organic matter in proglacial streams. Overall, our findings shed new light on benthic biofilms in proglacial streams now changing at rapid pace owing to climate-induced glacier shrinkage.

Microdiversity characterizes prevalent phylogenetic clades in the glacier-fed stream microbiome.

Glacier-fed streams (GFSs) are extreme and rapidly vanishing ecosystems, and yet they harbor diverse microbial communities. Although our understanding of the GFS microbiome has recently increased, we do not know which microbial clades are ecologically successful in these ecosystems, nor do we understand potentially underlying mechanisms. Ecologically successful clades should be more prevalent across GFSs compared to other clades, which should be reflected as clade-wise distinctly low phylogenetic turnover. However, methods to assess such patterns are currently missing. Here we developed and applied a novel analytical framework, "phyloscore analysis", to identify clades with lower spatial phylogenetic turnover than other clades in the sediment microbiome across twenty GFSs in New Zealand. These clades constituted up to 44% and 64% of community α-diversity and abundance, respectively. Furthermore, both their α-diversity and abundance increased as sediment chlorophyll a decreased, corroborating their ecological success in GFS habitats largely devoid of primary production. These clades also contained elevated levels of putative microdiversity than others, which could potentially explain their high prevalence in GFSs. This hitherto unknown microdiversity may be threatened as glaciers shrink, urging towards further genomic and functional exploration of the GFS microbiome.

An integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method to simultaneously quantify the infectious concentrations of eight environmentally relevant enterovirus serotypes.

Enterovirus (EV) infectivity is typically measured as a bulk parameter, yet EV serotypes vary in their susceptibility to natural and engineered stressors. Here we developed an integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method to simultaneously and specifically quantify the infectious concentrations of eight EV serotypes commonly encountered in sewage (coxsackieviruses A9, B1, B2, B3, B4 and B5, and echoviruses 25 and 30). The method uses two cell lines for virus replication and serotype-specific qPCR primers for quantification. Primers were designed to target multiple environmental strains of a given serotype and displayed high specificity. The ICC-RTqPCR method exhibited a linear calibration range between 50 and 1000 (echoviruses) or 5000 (coxsackieviruses) infectious units per mL. Over this range, measurements were not influenced by the presence of non-target serotypes, and calibration slopes were reproducible for different virus batches and cell ages. The ICC-RTqPCR method was able to accurately quantify the infectious concentration of a virus after inactivation by heat, and the concentration of a virus within a wastewater matrix. This method will be valuable to assess the differing fates of EV serotypes in natural or engineered systems, and to portray the associated changes in EV population composition.

Optimised biomolecular extraction for metagenomic analysis of microbial biofilms from high-mountain streams.

Glacier-fed streams (GFS) are harsh ecosystems dominated by microbial life organized in benthic biofilms, yet the biodiversity and ecosystem functions provided by these communities remain under-appreciated. To better understand the microbial processes and communities contributing to GFS ecosystems, it is necessary to leverage high throughput sequencing. Low biomass and high inorganic particle load in GFS sediment samples may affect nucleic acid extraction efficiency using extraction methods tailored to other extreme environments such as deep-sea sediments. Here, we benchmarked the utility and efficacy of four extraction protocols, including an up-scaled phenol-chloroform protocol. We found that established protocols for comparable sample types consistently failed to yield sufficient high-quality DNA, delineating the extreme character of GFS. The methods differed in the success of downstream applications such as library preparation and sequencing. An adapted phenol-chloroform-based extraction method resulted in higher yields and better recovered the expected taxonomic profile and abundance of reconstructed genomes when compared to commercially-available methods. Affordable and straight-forward, this method consistently recapitulated the abundance and genomes of a mock community, including eukaryotes. Moreover, by increasing the amount of input sediment, the protocol is readily adjustable to the microbial load of the processed samples without compromising protocol efficiency. Our study provides a first systematic and extensive analysis of the different options for extraction of nucleic acids from glacier-fed streams for high-throughput sequencing applications, which may be applied to other extreme environments.

Benchmarking protocols for the metagenomic analysis of stream biofilm viromes.

Viruses drive microbial diversity, function and evolution and influence important biogeochemical cycles in aquatic ecosystems. Despite their relevance, we currently lack an understanding of their potential impacts on stream biofilm structure and function. This is surprising given the critical role of biofilms for stream ecosystem processes. Currently, the study of viruses in stream biofilms is hindered by the lack of an optimized protocol for their extraction, concentration and purification. Here, we evaluate a range of methods to separate viral particles from stream biofilms, and to concentrate and purify them prior to DNA extraction and metagenome sequencing. Based on epifluorescence microscopy counts of viral-like particles (VLP) and DNA yields, we optimize a protocol including treatment with tetrasodium pyrophosphate and ultra-sonication to disintegrate biofilms, tangential-flow filtration to extract and concentrate VLP, followed by ultracentrifugation in a sucrose density gradient to isolate VLP from the biofilm slurry. Viromes derived from biofilms sampled from three different streams were dominated by Siphoviridae, Myoviridae and Podoviridae and provide first insights into the viral diversity of stream biofilms. Our protocol optimization provides an important step towards a better understanding of the ecological role of viruses in stream biofilms.