Multiple stressors alter greenhouse gas concentrations in streams through local and distal processes.

Streams are significant contributors of greenhouse gases (GHG) to the atmosphere, and the increasing number of stressors degrading freshwaters may exacerbate this process, posing a threat to climatic stability. However, it is unclear whether the influence of multiple stressors on GHG concentrations in streams results from increases of in-situ metabolism (i.e., local processes) or from changes in upstream and terrestrial GHG production (i.e., distal processes). Here, we hypothesize that the mechanisms controlling multiple stressor effects vary between carbon dioxide (CO2) and methane (CH4), with the latter being more influenced by changes in local stream metabolism, and the former mainly responding to distal processes. To test this hypothesis, we measured stream metabolism and the concentrations of CO2 (pCO2) and CH4 (pCH4) in 50 stream sites that encompass gradients of nutrient enrichment, oxygen depletion, thermal stress, riparian degradation and discharge. Our results indicate that these stressors had additive effects on stream metabolism and GHG concentrations, with stressor interactions explaining limited variance. Nutrient enrichment was associated with higher stream heterotrophy and pCO2, whereas pCH4 increased with oxygen depletion and water temperature. Discharge was positively linked to primary production, respiration and heterotrophy but correlated negatively with pCO2. Our models indicate that CO2-equivalent concentrations can more than double in streams that experience high nutrient enrichment and oxygen depletion, compared to those with oligotrophic and oxic conditions. Structural equation models revealed that the effects of nutrient enrichment and discharge on pCO2 were related to distal processes rather than local metabolism. In contrast, pCH4 responses to nutrient enrichment, discharge and temperature were related to both local metabolism and distal processes. Collectively, our study illustrates potential climatic feedbacks resulting from freshwater degradation and provides insight into the processes mediating stressor impacts on the production of GHG in streams.

Os rios são grandes emissores de gases com efeito de estufa (GEE) para a atmosfera, e o crescente número de agentes de stress que degradam os rios pode exacerbar este processo, e constituir uma ameaça à estabilidade climática. No entanto, não é claro se o efeito dos impactos humanos nas concentrações de GEE na água está associado ao aumento do metabolismo local do rio (processos locais) ou ao aumento da produção de GEE nas zonas a montante dos rios ou nas zonas terrestres adjacentes (processos distais). A nossa hipótese é que os mecanismos que controlam os efeitos dos impactos humanos na emissão de GEE variam entre o dióxido de carbono (CO2) e o metano (CH4). A nossa previsão é que o CO2 responde principalmente a processos distais, enquanto o CH4 é mais influenciado por alterações no metabolismo local dos cursos de água. Para avaliar esta hipótese, medimos o metabolismo aquático e as concentrações de CO2 (pCO2) e CH4 (pCH4) em 50 rios que abrangem gradientes de enriquecimento em nutrientes, depleção de oxigénio, stress térmico, degradação da zona ribeirinha e caudal. Os nossos resultados indicam que estes agentes de stress tiveram efeitos aditivos no metabolismo e nas concentrações de GEE nos rios, e que as interações entre os agentes de stress tiveram pouca capacidade preditiva. O enriquecimento em nutrientes foi associado a um aumento da heterotrofia e pCO2, enquanto o pCH4 aumentou com a depleção de oxigénio e com a temperatura da água. O caudal estava positivamente correlacionado com a produção primária, a respiração e a heterotrofia, mas negativamente correlacionado com o pCO2. Os nossos modelos indicam que as concentrações equivalentes de CO2 podem duplicar em rios eutrofizados e com baixa concentração de oxigénio, em comparação com os rios oligotróficos e com águas bem oxigenadas. A aplicação de modelos de equações estruturais mostrou que os efeitos do enriquecimento em nutrientes e do caudal no pCO2 estavam relacionados com processos distais e não com o metabolismo local. Em contrapartida, as respostas do pCH4 ao enriquecimento de nutrientes, ao caudal e à temperatura estavam relacionadas tanto com o metabolismo local como com processos distais. O nosso estudo demonstra que a degradação dos rios e dos ecossistemas ribeirinhos pode ter efeitos negativos na estabilidade climática e fornece informação relevante sobre os processos biogeoquímicos que medeiam os impactos humanos na produção de GEE nos rios.

Land Cover Implications on Ecosystem Service Delivery: a Multi-Scenario Study of Trade-offs and Synergies in River Basins.

Land cover change scenarios hold far-reaching implications for ecosystem services (ES), highlighting the need for understanding the trade-offs and synergies underlying the provision of multiple ES. The insufficient knowledge of the mechanisms governing the relationships among multiple ES, along with the lack of information on trade-offs among ES under different scenarios, restricts the ability to provide effective information for decision-makers. To fill this gap, we assessed the interplay among six ES: climate regulation, habitat creating and maintaining species diversity, cultivated crops, regulation of the chemical condition of freshwaters by living processes (water quality), water yield, and control of erosion rates, within three river basins in northwest Portugal. We employed the InVEST to map the state of these ES in 2018, along with three projected land cover scenarios for 2050: business-as-usual, farmland return, and afforestation. Our findings indicated the business-as-usual scenario could lead to detrimental impacts on climate regulation, habitat creating and maintaining species diversity, and control of erosion rates. In contrast, the farmland return scenario showed less drastic decreases in habitat-creating and maintaining species diversity and control of erosion rates compared to the business-as-usual scenario. Afforestation emerged as the most favorable scenario, with a 13.6% increase in climate regulation and a 1.3% improvement in habitat-creating and maintaining species diversity. Cluster analysis allowed the identification of six levels of spatial synergies between ES, with regions of high forest cover showing extreme synergy and populated areas exhibiting the lowest levels of synergy, suggesting that a well-planned combination of these practices could yield substantial benefits for future ES provision. These results provide crucial insights for decision-makers to enhance ecosystem management and promote societal well-being. Importantly, our findings underscore the significance of considering multiple ES and their interrelationships in land use planning to achieve sustainable development objectives.

Eco-physiological Responses of Aquatic Fungi to Three Global Change Stressors Highlight the Importance of Intraspecific Trait Variability.

Anthropogenic change at a global scale is affecting life on Earth with impacts on freshwaters. Aquatic hyphomycetes are fungi that drive organic matter decomposition in freshwaters and mediate energy transfer to higher trophic levels. Intraspecific trait variability affects ecological processes and can account for species adaptations to environmental change. To ascertain how aquatic hyphomycetes respond to global change related stressors, we selected 20 strains (7 species), based on their co-occurrence in streams and phylogenetic relatedness. We measured fungal growth rates at different temperatures (7 levels), nutrient concentrations (6 levels) and medium moisture (6 levels). Our results indicate that all stressors affected fungal growth, and responses to nutrient enrichment and moisture were strain specific. Fungal responses to the stressors were not explained by their phylogenetic relatedness. In the absence of stressors, interspecific diversity best explained the variance in fungal traits, while the increase in the stress gradient increased the importance of intraspecific diversity.

Elevated temperature may reduce functional but not taxonomic diversity of fungal assemblages on decomposing leaf litter in streams.

Mounting evidence points to a linkage between biodiversity and ecosystem functioning (B-EF). Global drivers, such as warming and nutrient enrichment, can alter species richness and composition of aquatic fungal assemblages associated with leaf-litter decomposition, a key ecosystem process in headwater streams. However, effects of biodiversity changes on ecosystem functions might be countered by the presumed high functional redundancy of fungal species. Here, we examined how environmental variables and leaf-litter traits (based on leaf chemistry) affect taxonomic and functional α- and ß-diversity of fungal decomposers. We analysed taxonomic diversity (DNA-fingerprinting profiles) and functional diversity (community-level physiological profiles) of fungal communities in four leaf-litter species from four subregions differing in stream-water characteristics and riparian vegetation. We hypothesized that increasing stream-water temperature and nutrients would alter taxonomic diversity more than functional diversity due to the functional redundancy among aquatic fungi. Contrary to our expectations, fungal taxonomic diversity varied little with stream-water characteristics across subregions, and instead taxon replacement occurred. Overall taxonomic ß-diversity was fourfold higher than functional diversity, suggesting a high degree of functional redundancy among aquatic fungi. Elevated temperature appeared to boost assemblage uniqueness by increasing ß-diversity while the increase in nutrient concentrations appeared to homogenize fungal assemblages. Functional richness showed a negative relationship with temperature. Nonetheless, a positive relationship between leaf-litter decomposition and functional richness suggests higher carbon use efficiency of fungal communities in cold waters.

Individual and mixed effects of anticancer drugs on freshwater rotifers: A multigenerational approach.

Human population growth has led to an increased release of chemical contaminants into aquatic environments. Emerging chemical contaminants (ECCs) are of increasing concern because they can affect non-target organisms in aquatic ecosystems. The application of anticancer drugs is increasing because of enhanced cancer rates and use of chemotherapy. We assessed the impacts of two widely used anticancer drugs known for their distinct modes of action, namely 5-fluorouracil (5-FU) and doxorubicin (DOX), on the freshwater rotifer Brachionus calyciflorus across generations. Rotifer mortality (24 h) and population growth (48 h) were assessed to determine initial lethal and sub-lethal effects. Exposure of rotifers to 5-FU (up to 200 mg L-1) did not cause mortality, while DOX caused mortality at high concentrations (EC50 = 15.6 mg L-1). Effects of 5-FU on population growth rate was higher than DOX (5-FU EC50 =10.49 µg L-1, DOX EC50 = 8.78 mg L-1). The effects of the drugs in binary mixture on population growth rates were dose dependent; significant antagonistic effects were found when 5-FU was present in the mixture at high concentrations. Finally, a transgenerational assay for five generations revealed that rotifers were able to recover their population growth rate after fourth generation when exposed to 5-FU; however, population became non-viable after the second generation of exposure to DOX. At the cellular level, accumulation of reactive oxygen species and plasma membrane damage were observed at EC10 and increased at EC50 for both drugs. After exposure of rotifers to 5-FU across generations, there were signs of oxidative stress recovery, as shown by a decrease in ROS accumulation and plasma membrane damage. Our results showed for the first time that the adverse effects of anticancer drugs on freshwater rotifer populations are drug and dose dependent and can persist or be attenuated along generations.

How do physicochemical properties influence the toxicity of silver nanoparticles on freshwater decomposers of plant litter in streams?

AgNP physicochemical properties may affect AgNP toxicity, but their effects on plant litter decomposition and the species driving this key ecosystem process in freshwaters have been poorly investigated. We assessed the impacts of AgNPs with different size and surface coating (100nm PVP (polyvinylpyrrolidone)-dispersant, 50-60nm and 35nm uncoated) on freshwater decomposers of leaf litter by exposing leaf associated microbial assemblages to increasing concentrations of AgNPs (up to 200mgL-1) and of AgNO3 (up to 25mgL-1). We further conducted a feeding preference experiment with a common invertebrate shredder, Limnephilus sp., which was allowed to feed on microbially-colonized leaves previously exposed to AgNPs and AgNO3. Leaf decomposition and microbial activity and diversity were inhibited when exposed to increased concentrations of 100nm AgNPs (≥25mgL-1), while microbial activity was stimulated by exposure to 35nm AgNPs (≥100mgL-1). Invertebrate shredders preferred leaves exposed to 35nm AgNPs (25mgL-1) and avoided leaves exposed to AgNO3 (≥2mgL-1). Results from the characterization of AgNPs by dynamic light scattering revealed that AgNps with PVP-dispersant were more stable than the uncoated AgNPs. Our results highlight the importance of considering the physicochemical properties of NPs when assessing their toxicity to litter decomposers in freshwaters.

Seasonal Variability May Affect Microbial Decomposers and Leaf Decomposition More Than Warming in Streams.

Ongoing climate change is expected to affect the diversity and activity of aquatic microbes, which play a key role in plant litter decomposition in forest streams. We used a before-after control-impact (BACI) design to study the effects of warming on a forest stream reach. The stream reach was divided by a longitudinal barrier, and during 1 year (ambient year) both stream halves were at ambient temperature, while in the second year (warmed year) the temperature in one stream half was increased by ca. 3 °C above ambient temperature (experimental half). Fine-mesh bags containing oak (Quercus robur L.) leaves were immersed in both stream halves for up to 60 days in spring and autumn of the ambient and warmed years. We assessed leaf-associated microbial diversity by denaturing gradient gel electrophoresis and identification of fungal conidial morphotypes and microbial activity by quantifying leaf mass loss and productivity of fungi and bacteria. In the ambient year, no differences were found in leaf decomposition rates and microbial productivities either between seasons or stream halves. In the warmed year, phosphorus concentration in the stream water, leaf decomposition rates, and productivity of bacteria were higher in spring than in autumn. They did not differ between stream halves, except for leaf decomposition, which was higher in the experimental half in spring. Fungal and bacterial communities differed between seasons in both years. Seasonal changes in stream water variables had a greater impact on the activity and diversity of microbial decomposers than a warming regime simulating a predicted global warming scenario.

Polyhydroxyfullerene binds cadmium ions and alleviates metal-induced oxidative stress in Saccharomyces cerevisiae.

The water-soluble polyhydroxyfullerene (PHF) is a functionalized carbon nanomaterial with several industrial and commercial applications. There have been controversial reports on the toxicity and/or antioxidant properties of fullerenes and their derivatives. Conversely, metals have been recognized as toxic mainly due to their ability to induce oxidative stress in living organisms. We investigated the interactive effects of PHF and cadmium ions (Cd) on the model yeast Saccharomyces cerevisiae by exposing cells to Cd (≤5 mg liter(-1)) in the absence or presence of PHF (≤500 mg liter(-1)) at different pHs (5.8 to 6.8). In the absence of Cd, PHF stimulated yeast growth up to 10.4%. Cd inhibited growth up to 79.7%, induced intracellular accumulation of reactive oxygen species (ROS), and promoted plasma membrane disruption in a dose- and pH-dependent manner. The negative effects of Cd on growth were attenuated by the presence of PHF, and maximum growth recovery (53.8%) was obtained at the highest PHF concentration and pH. The coexposure to Cd and PHF decreased ROS accumulation up to 36.7% and membrane disruption up to 30.7% in a dose- and pH-dependent manner. Two mechanisms helped to explain the role of PHF in alleviating Cd toxicity to yeasts: PHF decreased Cd-induced oxidative stress and bound significant amounts of Cd in the extracellular medium, reducing its bioavailability to the cells.

Fungal identity mediates the impacts of multiple stressors on freshwater ecosystems.

Predicting how multiple anthropogenic stressors affect natural ecosystems is a major challenge in ecology. Freshwater ecosystems are threatened worldwide by multiple co-occurring stressors, which can affect aquatic biodiversity, ecosystem functioning and human wellbeing. In stream ecosystems, aquatic fungi play a crucial role in global biogeochemical cycles and food web dynamics, therefore, assessing the functional consequences of fungal biodiversity loss under multiple stressors is crucial. Here, a microcosm approach was used to investigate the effects of multiple stressors (increased temperature and nutrients, drying, and biodiversity loss) on three ecosystem processes: organic matter decomposition, fungal reproduction, and fungal biomass accrual. Net effects of stressors were antagonistic for organic matter decomposition, but additive for fungal reproduction and biomass accrual. Net effects of biodiversity were mainly positive for all processes, even under stress, demonstrating that diversity assures the maintenance of ecosystem processes. Fungal species displayed distinct contributions to each ecosystem process. Furthermore, species with negligible contributions under control conditions changed their role under stress, either enhancing or impairing the communities' performance, emphasizing the importance of fungal species identity. Our study highlights that distinct fungal species have different sensitivities to environmental variability and have different influence on the overall performance of the community. Therefore, preserving high fungal diversity is crucial to maintain fungal species with key ecosystem functions within aquatic communities in face of environmental change.

Current trends and mismatches on fungicide use and assessment of the ecological effects in freshwater ecosystems.

Despite increasing evidence of off-site ecological impacts of pesticides and policy efforts worldwide, pesticide use is still far from being ecologically sustainable. Fungicides are among the most sold classes of pesticides and are crucial to ensure global food supply and security. This study aimed to identify potential gaps of knowledge and mismatches between research and usage data of fungicides by: (i) systematizing the current trends in global sales of fungicides, focusing on the European context in particular (where they are proportionally important); (ii) reviewing the scientific literature on the impacts of synthetic fungicides on non-target freshwater organisms. Sales data revealed important global and regional asymmetries in the relative importance of fungicides and the preferred active ingredients. The literature review on the ecological effects of fungicides disclosed a mismatch between the most studied and the most sold substances, as well as a bias towards the use of single species assays with standard test organisms. To ensure a proper evaluation, risk scenarios should focus on a regional scale, and research agendas must highlight sensitive aquatic ecorreceptors and improve the crosstalk between analytical and sales data.

Effects of riparian plant diversity loss on aquatic microbial decomposers become more pronounced with increasing time.

We examined the potential long-term impacts of riparian plant diversity loss on diversity and activity of aquatic microbial decomposers. Microbial assemblages were obtained in a mixed-forest stream by immersion of mesh bags containing three leaf species (alder, oak and eucalyptus), commonly found in riparian corridors of Iberian streams. Simulation of species loss was done in microcosms by including a set of all leaf species, retrieved from the stream, and non-colonized leaves of three, two or one leaf species. Leaves were renewed every month throughout six months, and microbial inoculum was ensured by a set of colonized leaves from the previous month. Microbial diversity, leaf mass loss and fungal biomass were assessed at the second and sixth months after plant species loss. Molecular diversity of fungi and bacteria, as the total number of operational taxonomic units per leaf diversity treatment, decreased with leaf diversity loss. Fungal biomass tended to decrease linearly with leaf species loss on oak and eucalyptus, suggesting more pronounced effects of leaf diversity on lower quality leaves. Decomposition of alder and eucalyptus leaves was affected by leaf species identity, mainly after longer times following diversity loss. Leaf decomposition of alder decreased when mixed with eucalyptus, while decomposition of eucalyptus decreased in mixtures with oak. Results suggest that the effects of leaf diversity on microbial decomposers depended on leaf species number and also on which species were lost from the system, especially after longer times. This may have implications for the management of riparian forests to maintain stream ecosystem functioning.

Drying shapes the ecological niche of aquatic fungi with implications on ecosystem functioning.

Fungi are among the most abundant and diverse organisms on Earth and play pivotal roles in global carbon processing, nutrient cycling and food webs. Despite their abundant and functional importance, little is known about the patterns and mechanisms governing their community composition in intermittent rivers and ephemeral streams, which are the most common fluvial ecosystems globally. Thus far, it is known that aquatic fungi have evolved various life-history strategies and functional adaptations to cope with drying. Nevertheless, some of these adaptations have a metabolic cost and trade-offs between growth, reproduction and dispersion that may affect ecosystem functioning. Thus, understanding their ecological strategies along a gradient of drying is crucial to assess how species will respond to global change and to identify meaningful taxa to maintain ecosystem functions. By combining in situ hydrological information with a niche-based approach, we analysed the role of drying in explaining the spatial segregation of fungal species, and we determined their specialization and affinity over a gradient of drying. In addition, we estimated whether species niches are good predictors of two key ecosystem processes: organic matter decomposition and fungal biomass accrual. Overall, we found that annual drying duration and frequency were the most influential variables upon species niche differentiation across the 15 studied streams. Our cluster analysis identified four drying niche-based groups with contrasting distributions and responses over the drying gradient: drying-sensitive, partly tolerant to drying, generalist, and drying-resistant specialist. In addition, we found that species belonging to the drying specialist group showed a weak contribution to both ecosystem processes, suggesting trade-offs between drying resistance strategies and the energy invested in growth. Taken together, our results suggest that increased water scarcity may jeopardise the capacity of aquatic fungi to guarantee ecosystem functioning and to maintain biogeochemical cycles despite their ability to cope with drying.

Extreme weather events threaten biodiversity and functions of river ecosystems: evidence from a meta-analysis.

Both gradual and extreme weather changes trigger complex ecological responses in river ecosystems. It is still unclear to what extent trend or event effects alter biodiversity and functioning in river ecosystems, adding considerable uncertainty to predictions of their future dynamics. Using a comprehensive database of 71 published studies, we show that event - but not trend - effects associated with extreme changes in water flow and temperature substantially reduce species richness. Furthermore, event effects - particularly those affecting hydrological dynamics - on biodiversity and primary productivity were twice as high as impacts due to gradual changes. The synthesis of the available evidence reveals that event effects induce regime shifts in river ecosystems, particularly affecting organisms such as invertebrates. Among extreme weather events, dryness associated with flow interruption caused the largest effects on biota and ecosystem functions in rivers. Effects on ecosystem functions (primary production, organic matter decomposition and respiration) were asymmetric, with only primary production exhibiting a negative response to extreme weather events. Our meta-analysis highlights the disproportionate impact of event effects on river biodiversity and ecosystem functions, with implications for the long-term conservation and management of river ecosystems. However, few studies were available from tropical areas, and our conclusions therefore remain largely limited to temperate river systems. Further efforts need to be directed to assemble evidence of extreme events on river biodiversity and functioning.

Evidence of micro and macroplastic toxicity along a stream detrital food-chain.

Freshwater ecosystems are subjected to plastic extensive pollution because they are the direct link between plastic wastes and marine ecosystems. The aim of this study was to assess the impacts of different sizes of polyethylene plastics (micro: µPs and macroplastics: PBs) on freshwater decomposers of plant litter. We exposed leaf associated microbial assemblages to µPs (0.5 or 1.5 g L-1) and discs of PBs as follows: green plastic bags (PB-G) alone or in mixtures with transparent plastic bags (PB-Mix). Then, we conducted a feeding preference experiment with the invertebrate shredder Limnephilus sp. to assess their capacity to distinguish leaf discs from PB discs of the same size (12 mm). Leaf decomposition, activities of fungal enzymes and sporulation were inhibited by µPs and PB-Mix, and shifts in fungal community composition were observed. The invertebrate shredders preferred to feed on leaves treated with µPs avoiding those exposed to PB-G/PB-Mix. Our results demonstrated that plastics can have a direct effect on stream-dwelling microbial decomposers and an indirect effect on higher trophic levels (shredders), highlighting that trophic transfer is a route of plastic exposure. The plastic properties (size, concentration, colour) appear to influence plastic toxicity to microbes and shredders, indicating the importance of considering physicochemical properties when assessing their risks to freshwater ecosystems.

Can microplastics from personal care products affect stream microbial decomposers in the presence of silver nanoparticles?

Microplastics (MPs) are emerging contaminants of great concern due to their abundance and persistence over time in aquatic environments. However, studies on their impacts on freshwater organisms are scarce. In resemblance, silver nanoparticles (Ag-NPs) are incorporated into textiles and personal care products and are also classified as emerging contaminants. We used the leaf litter decomposition model system to investigate the effects of MPs from a commercially used personal care product, alone or in mixture with Ag-NPs, on the diversity and activities of freshwater microbial decomposers. We exposed stream microbial communities associated with leaf litter to increasing concentrations of MPs (polyethylene extracted from a personal care product; 100 µg L-1 up to 1 g L-1 5 concentrations plus 1 control) for 27 days in the absence or presence of Ag-NPs (0.1 mg L-1 and 1 mg L-1). The exposure to MPs, alone or in mixture with Ag-NPs, negatively affected fungal diversity and sporulation, with a reduction in leaf litter decomposition (Cohen's d > 1.5; r> 0.8; Bonferroni, P < 0.01). Shifts in community structure of sporulating fungi were observed, and effects were more pronounced in mixtures with Ag-NPs at the highest concentration. Mixtures of MPs with Ag-NPs (at the higher concentration) had the strongest impacts on extracellular enzymatic (ß-glucosidase, Cohen's d > 1; r > 0.5; phenol oxidase, Cohen's d > 1; r > 0.4) activities (ANOVAs, P < 0.05). Apart from sporulation rates, observed toxicity in mixtures was lower than that expected based on individual toxicity effects, mainly for higher concentrations (Bonferroni, P < 0.05). Our study provided evidence of the potential harmful effects of MPs, alone or in mixtures with Ag-NPs, on the activities of aquatic fungi and on a key ecosystem process, determinant to organic matter turnover in streams.

ITS rDNA Barcodes Clarify Molecular Diversity of Aquatic Hyphomycetes.

Aquatic hyphomycetes are key microbial decomposers of allochthonous organic matter in freshwater ecosystems. Although their importance in carbon flow and food webs in streams is widely recognized, there are still gaps in our understanding of their molecular diversity and distribution patterns. Our study utilized the growing database of ITS rDNA barcodes of aquatic hyphomycetes (1252 sequences) and aimed to (i) produce new barcodes for some lesser-known taxa; (ii) clarify the taxonomic placement of some taxa at the class or order level, based on molecular data; and (iii) provide insights into the biogeographical origins of some taxa. This study increased the number of aquatic hyphomycete species with available ITS barcodes from 119 (out of ~300 species described) to 136. Phylogenetically, the 136 species were distributed between 2 phyla, 6 classes, and 10 orders of fungi. Future studies should strive to increase the database of ITS sequences, especially focusing on species with unclear phylogenetic relationships (incertae sedis) and with few sequences available. The geographical distribution of species with available ITS sequences included 50 countries from five continents, but 6 countries had more than 20 species associated, showing a bias toward the northern hemisphere, likely due to sampling bias.

Can metal nanoparticles be a threat to microbial decomposers of plant litter in streams?

The extensive use of nanometal-based products increases the chance of their release into aquatic environments, raising the question whether they can pose a risk to aquatic biota and the associated ecological processes. Aquatic microbes, namely fungi and bacteria, play a key role in forested streams by decomposing plant litter from terrestrial vegetation. Here, we investigated the effects of nanocopper oxide and nanosilver on leaf litter decomposition by aquatic microbes, and the results were compared with the impacts of their ionic precursors. Alder leaves were immersed in a stream of Northwest Portugal to allow microbial colonization before being exposed in microcosms to increased nominal concentrations of nanometals (CuO, 100, 200 and 500 ppm; Ag, 100 and 300 ppm) and ionic metals (Cu(2+) in CuCl(2), 10, 20 and 30 ppm; Ag(+) in AgNO(3), 5 and 20 ppm) for 21 days. Results showed that rates of leaf decomposition decreased with exposure to nano- and ionic metals. Nano- and ionic metals inhibited bacterial biomass (from 68.6% to 96.5% of control) more than fungal biomass (from 28.5% to 82.9% of control). The exposure to increased concentrations of nano- and ionic metals decreased fungal sporulation rates from 91.0% to 99.4%. These effects were accompanied by shifts in the structure of fungal and bacterial communities based on DNA fingerprints and fungal spore morphology. The impacts of metal nanoparticles on leaf decomposition by aquatic microbes were less pronounced compared to their ionic forms, despite metal ions were applied at one order of magnitude lower concentrations. Overall, results indicate that the increased release of nanometals to the environment may affect aquatic microbial communities with impacts on organic matter decomposition in streams.

Intraspecific traits change biodiversity effects on ecosystem functioning under metal stress.

Studies investigating the impacts of biodiversity loss on ecosystem processes have often reached different conclusions, probably because insufficient attention has been paid to some aspects including (1) which biodiversity measure (e.g., species number, species identity or trait) better explains ecosystem functioning, (2) the mechanisms underpinning biodiversity effects, and (3) how can environmental context modulates biodiversity effects. Here, we investigated how species number (one to three species) and traits of aquatic fungal decomposers (by replacement of a functional type from an unpolluted site by another from a metal-polluted site) affect fungal production (biomass accumulation) and plant litter decomposition in the presence and absence of metal stress. To examine the putative mechanisms that explain biodiversity effects, we determined the contribution of each fungal species to the total biomass produced in multicultures by real-time PCR. In the absence of metal, positive diversity effects were observed for fungal production and leaf decomposition as a result of species complementarity. Metal stress decreased diversity effects on leaf decomposition in assemblages containing the functional type from the unpolluted site, probably due to competitive interactions between fungi. However, dominance effect maintained positive diversity effects under metal stress in assemblages containing the functional type from the metal-polluted site. These findings emphasize the importance of intraspecific diversity in modulating diversity effects under metal stress, providing evidence that trait-based diversity measures should be incorporated when examining biodiversity effects.

Effects of cadmium and phenanthrene mixtures on aquatic fungi and microbially mediated leaf litter decomposition.

Urbanization and industrial activities have contributed to widespread contamination by metals and polycyclic aromatic hydrocarbons, but the combined effects of these toxics on aquatic biota and processes are poorly understood. We examined the effects of cadmium (Cd) and phenanthrene on the activity and diversity of fungi associated with decomposing leaf litter in streams. Leaves of Alnus glutinosa were immersed for 10 days in an unpolluted low-order stream in northwest Portugal to allow microbial colonization. Leaves were then exposed in microcosms for 14 days to Cd (0.06-4.5 mg L(-1)) and phenanthrene (0.2 mg L(-1)) either alone or in mixture. A total of 19 aquatic hyphomycete species were found sporulating on leaves during the whole study. The dominant species was Articulospora tetracladia, followed by Alatospora pulchella, Clavatospora longibrachiata, and Tetrachaetum elegans. Exposure to Cd and phenanthrene decreased the contribution of A. tetracladia to the total conidial production, whereas it increased that of A. pulchella. Fungal diversity, assessed as denaturing gradient gel electrophoresis fingerprinting or conidial morphology, was decreased by the exposure to Cd and/or phenanthrene. Moreover, increased Cd concentrations decreased leaf decomposition and fungal reproduction but did not inhibit fungal biomass production. Exposure to phenanthrene potentiated the negative effects of Cd on fungal diversity and activity, suggesting that the co-occurrence of these stressors may pose additional risk to aquatic biodiversity and stream ecosystem functioning.

Transcriptomics reveals the action mechanisms and cellular targets of citrate-coated silver nanoparticles in a ubiquitous aquatic fungus.

Silver nanoparticles (AgNPs) are among the major groups of contaminants of emerging concern for aquatic ecosystems. The massive application of AgNPs relies on the antimicrobial properties of Ag, raising concerns about their potential risk to ecologically important freshwater microbes and the processes they drive. Moreover, it is still uncertain whether the effects of AgNPs are driven by the same mechanisms underlying those of Ag ions (Ag+). We employed transcriptomics to better understand AgNP toxicity and disentangle the role of Ag+ in the overall toxicity towards aquatic fungi. To that end, the worldwide-distributed aquatic fungus Articulospora tetracladia, that plays a central role in organic matter turnover in freshwaters, was selected and exposed for 3 days to citrate-coated AgNPs (∼20 nm) and Ag+ at concentrations inhibiting 20% of growth (EC20). Responses revealed 258 up- and 162 down-regulated genes upon exposure to AgNPs and 448 up- and 84 down-regulated genes under exposure to Ag+. Different gene expression patterns were found after exposure to each silver form, suggesting distinct mechanisms of action. Gene ontology (GO) analyses showed that the major cellular targets likely affected by both silver forms were the biological membranes. GO-based biological processes indicated that AgNPs up-regulated the genes involved in transport, nucleobase metabolism and energy production, but down-regulated those associated with redox and carbohydrate metabolism. Ag+ up-regulated the genes involved in carbohydrate and steroid metabolism, whereas genes involved in localization and transport were down-regulated. Our results showed, for the first time, distinct profiles of gene expression in aquatic fungi exposed to AgNPs and Ag+, supporting different modes of toxicity of each silver form. Also, our results suggest that Ag+ had a negligible role in the toxicity induced by AgNPs. Finally, our study highlights the power of transcriptomics in portraying the stress induced by different silver forms in organisms.