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.

Effects of warming on stream biofilm organic matter use capabilities.

The understanding of ecosystem responses to changing environmental conditions is becoming increasingly relevant in the context of global warming. Microbial biofilm communities in streams play a key role in organic matter cycling which might be modulated by shifts in flowing water temperature. In this study, we performed an experiment at the Candal stream (Portugal) longitudinally divided into two reaches: a control half and an experimental half where water temperature was 3 °C above that of the basal stream water. Biofilm colonization was monitored during 42 days in the two stream halves. Changes in biofilm function (extracellular enzyme activities and carbon substrate utilization profiles) as well as chlorophyll a and prokaryote densities were analyzed. The biofilm in the experimental half showed a higher capacity to decompose cellulose, hemicellulose, lignin, and peptidic compounds. Total leucine-aminopeptidase, cellobiohydrolase and ß-xylosidase showed a respective 93, 66, and 61% increase in activity over the control; much higher than would be predicted by only the direct temperature physical effect. In contrast, phosphatase and lipase activity showed the lowest sensitivity to temperature. The biofilms from the experimental half also showed a distinct functional fingerprint and higher carbon usage diversity and richness, especially due to a wider use of polymers and carbohydrates. The changes in the biofilm functional capabilities might be indirectly affected by the higher prokaryote and chlorophyll density measured in the biofilm of the experimental half. The present study provides evidence that a realistic stream temperature increase by 3 °C changes the biofilm metabolism to a greater decomposition of polymeric complex compounds and peptides but lower decomposition of lipids. This might affect stream organic matter cycling and the transfer of carbon to higher trophic levels.

Incubation temperature and substrate quality modulate sporulation by aquatic hyphomycetes.

Frequency and amplitude of temperature oscillations can profoundly affect structure and function of ecosystems. Unless the rate of a biological process changes linearly within the range of these fluctuations, the cumulative effect of temperature differs from the effect measured at the average temperature (Jensen's inequality). Here, we measured numbers and types of spores released by aquatic hyphomycetes from oak and alder leaves that had been exposed in a Portuguese stream for between 7 and 94 days. Recovered leaves were incubated at four temperatures between 5 and 20 °C. Over this range, the sporulation response to temperature was decelerating, with an estimated optimum around 12.5 °C. Assuming a linear response, therefore, overestimates spore release from decaying leaves. The calculated discrepancy was more pronounced with recalcitrant oak leaves (greater toughness, phenolics concentration, lower N and P concentration than alder), and reached 26.6 % when temperature was assumed to oscillate between 1 and 9 °C, rather than remaining constant at 5 °C. The maximum fluctuation of water temperature over 48 h during the field experiment was approximately 3 °C, which would result in a discrepancy of up to 6 %. The composition of the fungal community (assessed by species identification of released spores) was significantly influenced by the state of decomposition, but not by leaf species or temperature. When quantifying the potential impact of global change on aquatic fungal communities, the average increase as well as fluctuations of the temperature have to be considered.

Aquatic hyphomycete strains from metal-contaminated and reference streams might respond differently to future increase in temperature.

Aquatic hyphomycetes, a group of polyphyletic fungi, have been reported in streams contaminated with metals. This tolerance to metal contamination however can result in limited performance and limited ability to cope with additional environmental change. The predicted increase in water temperature, as a consequence of global warming, will have an additional effect on many streams. The sensitivity to temperature of strains of three aquatic hyphomycete species isolated from a metal-contaminated stream and an uncontaminated stream was assessed by determining their radial growth and activity (conidial production, oxygen consumption, mycelial biomass accumulation, fine particulate organic matter [FPOM] production, and microbial induced leaf mass loss) at 13 C (present water temperature in autumn) and at 18 C (predicted water temperature under global warming). Growth and reproductive activity generally were depressed for the strains isolated from the metal-contaminated stream when compared with those isolated from the unpolluted stream. These differences however were not translated into differences in FPOM production and leaf-litter mass loss, indicating that the strains isolated from the contaminated stream can decompose leaf litter similar to those of the reference stream. The 5 C increase in temperature stimulated fungal activity and litter decomposition, irrespective of species and strain. This might have strong effect on aquatic food-web and ecosystem functioning under global warming because increases in litter decomposition might lead to food shortage for higher trophic levels. The sensitivity to temperature depended on the response variable, species and strain. FPOM production was the variable most sensitive to temperature across strains and species and that for which temperature sensitivities differed most between strains. Fungal tolerance to metal contamination affects the extent to which its functions are stimulated by an increase in temperature, constituting an additional cost of metal tolerance.

Air temperature more than drought duration affects litter decomposition under flow intermittency.

Stream intermittency - periodic sequences of water flow cessation and resumption - occurs throughout the year, across seasons. Even though temperature is a known regulator of litter decomposition in both terrestrial and aquatic environments, comparative experiments on drought durations at distinct temperatures on microbial-mediated decomposition in streams experiencing intermittency are still lacking. Here, three drought temperatures (5, 15 and 25 °C) and two durations (short: 2.5 weeks; long: 5 weeks) were applied in a microcosm study to oak leaf discs colonized in a reference stream; mass loss and associated microbial parameters (fungal biomass, microbial activity, and sporulation rates) were evaluated following re-submersion for 2 weeks. Higher mass loss was found at 15 °C than 25 °C. A prolongation of the drought exposure period had no effect on mass loss, suggesting an early (≤ 2.5 weeks) inhibitor effect of drought on microbial-mediated leaf degradation. Fungal biomass was highest at 25 °C following a short drought, and decreased with a longer drought period at both 15 °C and 25 °C. Microbial activity was not affected by either drought duration or temperature. Sporulation rates and fungal diversity were significantly reduced by the longer drought period; in the short treatment, maximum values were found at 15 °C. In contrast to longer droughts, aquatic fungal communities during short dry periods seem to invest in energetically-expensive physiological responses to desiccation (e.g., ergosterol production) promoting biomass accrual at the expense of mass loss and reproductive output. Under more severe desiccation (higher duration and temperature), the lower diversity of fungal communities seem to result in negative legacy effects for fungal growth and reproductive capacity after flow resumption. These results suggest that native riparian vegetation, through its ability to regulate temperature in streams, may be critical in protecting freshwaters from intensified severity of drought periods in streams experiencing intermittency.

Salt pulses effects on in-stream litter processing and recovery capacity depend on substrata quality.

Human activities have greatly extended and intensified freshwater salinization, which threatens the structure and functioning of streams and rivers. Research on salt effects on in-stream processes has been strongly biased towards chronic salinization at constant levels. The aim of this study was to assess microbial mediated decomposition of two leaf species contrasting in quality (alder and oak) and associated descriptors, during salt-pulsed contamination (salinization period) and after cessation of salt additions (recovery period). Leaves were incubated in a mountain stream (central Portugal) longitudinally divided over 22 m. Half of the stream (salinized half) was subjected to daily short-term sharp salinity increases (conductivity up to ~48 mS cm-1) during 7 days while the other half (control half) was used as control. During the salinization period, salt exposure negatively affected mass loss and microbial respiration rate of alder (high-quality resource) while effects on fungal sporulation rate were independent of leaf quality. Fungal biomass was not impacted. After the recovery period, mass loss and respiration rate in both leaf species were similar between experimental stream halves. Fungal biomass associated with oak was enhanced and sporulation rate of alder, maintained in the previously salinized half, remained depressed. These results point out that the effects of salt pulses may be more deleterious in streams exclusively lined by high (vs. low) quality riparian trees as a result of a less efficient microbial-mediated leaf processing, and a reduced contribution to the conidial pool, even beyond the salinization period.

Contamination by uranium mine drainages affects fungal growth and interactions between fungal species and strains.

The presence of aquatic hyphomycetes has been reported for several heavy metal-contaminated waters. Tolerance probably is one adaptation to coping with heavy metals. To help clarify this issue strains of two species of aquatic hyphomycetes (Tricladium splendens Ingold and Varicosporium elodeae Kegel) were isolated from a reference stream and a stream contaminated with heavy metals and grown on malt extract agar prepared with reference and contaminated water to characterize colony morphology, growth rate, growth inhibition and interaction among species and strains. In V. elodeae the morphology of colonies differed between strains. Colony diameter increased linearly over time with growth rates being lower for strains isolated from contaminated than from reference streams (mostly for V. elodeae). Strains from the contaminated stream grew faster in medium prepared with contaminated water than in medium prepared with reference water, while for strains from the reference stream there was no significant difference in growth rates on the two media. In interacting isolates radial growth toward the opposing colony was generally lower than toward the dish edge. Percentage growth inhibition was higher for isolates in intraspecific interactions (13-37%) than in interspecific interactions (3-27%). However differences in growth inhibition experienced by interacting isolates were observed only in three cases out of 16. The difference between the percentage inhibition caused and experienced by a given isolate was highest in interactions involving isolates with distinct growth rates. Our results suggest that strains from the reference stream tolerate heavy metals while strains from the contaminated stream seem to be adapted to contaminated waters. We hypothesize that in natural environments fungal species-specific limits of tolerance to metal contamination might determine an abrupt or gradual response of the original fungal community to mine pollution giving origin to a poorer fungal community dominated by adapted strains with distinct functional efficiency.

Leaf litter microbial decomposition in salinized streams under intermittency.

Human-induced salinization of freshwaters constitutes a growing global problem, whose consequences on streams functioning are largely unknown. Climate change projections predict enhanced evaporation, as well as an increase in extreme events and in variability of precipitation. This will result in more frequent, extended and severe drought periods that may aggravate water salinization of streams and rivers. In this study we conducted a microcosm experiment to assess the combined effects of three drought regimes - abrupt (AD), slow (SD) and very slow transition to dryness (VSD) - and three levels of salinization (0, 4, 6 g L-1 NaCl) on microbial-mediated oak leaf decomposition over ten weeks. Salinization did not affect mass loss and associated microbial respiration of colonized oak leaves but significantly reduced the biomass and eliminated the sporulating capacity of fungi. Desiccation negatively affected leaf decomposition regardless of regime. Even though microbial respiration did not react to the different treatments, lower fungal biomass, diversity, and conidial production were observed under AD; for fungal biomass these effects were amplified at higher salt concentrations (particularly at 6 g L-1). Our results indicate that effects of leaf litter desiccation depend on the rate of transition between wet and dry conditions and on the level of salt in the water. The two factors jointly affect decomposer survival and activity and, by extension, the dynamics of detrital food webs in streams.

Does short-term salinization of freshwater alter the behaviour of the Iberian barbel (Luciobarbus bocagei, Steindachner 1864)?

Stream salinization is a great environmental hazard being aggravated by anthropogenic disturbances. Harmful conditions, as increasing salinity in freshwater systems, may negatively affect river fish fauna and possibly influence fish behaviour, such as boldness and/or cerebral lateralization. Salinity has been proven to affect behavioural expression, despite the tolerance of some species. It is thus relevant to study these behaviours, as the salinity exposure effects could represent greater environmental consequences. The impact of salinity stress was evaluated by exposing Iberian barbels, Luciobarbus bocagei (Steindachner, 1864) (Cypriniformes, Cyprinidae), to three levels of salinity (0.9, 9 and 19 mS/cm, using NaCl) and by conducting boldness and lateralization experiments, regarding population trends. Results show that, with increased salinity, fish diverged to the extremes of the shy-bold gradient, the population was slightly lateralized to the left, and seemed to become more lateralized with increasing salinity. However, there were no statistical differences between the treatments. Fish living in a Mediterranean climate are especially resilient to various stressors, which may confer them additional tolerance, and in this case, acute punctual exposure to increased salinity may not be detrimental for behaviour maintenance. We encourage the expansion of the research to different freshwater fish species that would help to recognise salinity thresholds and use them to implement effective conservation measures and appropriate ecological restoration actions for these sensible systems.

Global patterns and drivers of ecosystem functioning in rivers and riparian zones.

River ecosystems receive and process vast quantities of terrestrial organic carbon, the fate of which depends strongly on microbial activity. Variation in and controls of processing rates, however, are poorly characterized at the global scale. In response, we used a peer-sourced research network and a highly standardized carbon processing assay to conduct a global-scale field experiment in greater than 1000 river and riparian sites. We found that Earth's biomes have distinct carbon processing signatures. Slow processing is evident across latitudes, whereas rapid rates are restricted to lower latitudes. Both the mean rate and variability decline with latitude, suggesting temperature constraints toward the poles and greater roles for other environmental drivers (e.g., nutrient loading) toward the equator. These results and data set the stage for unprecedented "next-generation biomonitoring" by establishing baselines to help quantify environmental impacts to the functioning of ecosystems at a global scale.

Are fungal strains from salinized streams adapted to salt-rich conditions?

Anthropogenic salinization of freshwater is a global problem with largely unknown consequences for stream functions. We compared the effects of salt addition (6 g l-1 NaCl) in microcosms on leaf mass loss and microbial parameters in single- and multispecies assemblages of fungal strains (Heliscus lugdunensis, HELU; Tetracladium marchalianum, TEMA; Flagellospora curta, FLCU) isolated from a reference (R) or salinized (S) stream. Fungal growth and interactions were also assessed. Salinization inhibited leaf decomposition and fungal biomass, but no differences were observed between species, strains or species combinations. Sporulation rates in monocultures were not affected by added salt, but differed among species (FLCU > HELU > TEMA), with S strains releasing more conidia. Fungal assemblages did not differ significantly in total conidia production (either between strains or medium salt concentration). HELU was the dominant species, which also had highest growth and most pronounced antagonistic behaviour. Fungal species, irrespective of origin, largely maintained their function in salinized streams. Strains from salt-contaminated streams did not trade-off conidial production for vegetative growth at high salt levels. The expected reduction of fungal diversity and potential changes in nutritional litter quality owing to salinization may impact leaf incorporation into secondary production in streams.This article is part of the theme issue 'Salt in freshwaters: causes, ecological consequences and future prospects'.

Stream salinization and fungal-mediated leaf decomposition: A microcosm study.

Salinization is of major global concern due to its effect on stream biota, and ecosystem functions and services. In small streams, litter decomposition is a key ecosystem-level process driven by decomposers, mainly fungi (aquatic hyphomycetes), which link litter and invertebrates. Here we assessed the effects of an environmentally relevant range of salt additions (0, 2, 4, 8, 16gL-1 NaCl) on (1) fungal growth and species-specific reproductive output and (2) fungal mediated-decomposition of Quercus robur leaves. Growth rates of eight out of nine species of aquatic hyphomycetes were negatively affected by salinity at concentrations ≥4gL-1. EC50s were species-specific and ≥7.80gL-1. Distinct thresholds were observed for reproduction: only five species sporulated at 2gL-1, and a single one (Flagellospora curta) sporulated at 4 and 8gL-1 NaCl. Based on these results, we evaluated if tolerant fungal assemblages, with increasingly fewer species (9, 5, 1), were able to maintain similar functional functions and processes at the different salt levels. No significant differences were found in oak mass loss or sporulation rates at 0 or 2gL-1 NaCl; a clear inhibition of both parameters was observed at the highest concentrations (i.e., 4 and 8gL-1 NaCl). Different dominance patterns in multi-species fungal assemblages may determine bottom-up impacts on the stream food webs through effects on detritivore feeding preferences. Specific growth rate, characterized by RNA concentration, was higher in the single species, at the highest salt-concentration, and lower in the 9-species assemblage. Respiration was almost 2-times higher in mixed assemblages without added salt. Under salt-contamination, trade-offs between growth and sporulation seem to guarantee high levels of fungal growth and decomposition, particularly in multi-species assemblages. In the presence of salt contamination, aquatic hyphomycetes, even at reduced diversity, remain important drivers of leaf decomposition and ensure organic matter recycling.

Effects of Eucalyptus plantations on detritus, decomposers, and detritivores in streams.

Vast areas of the Iberian Peninsula are covered by monocultures of the exotic tree Eucalyptus globulus. Given that (1) leaf litter produced in the riparian areas is the main energy source for small streams, and (2) trees differ in their nutrient content, chemical defenses, and physical attributes, eucalypt plantations have the potential to affect the biology of streams. Research teams from the University of Coimbra and the University of the Basque Country have been addressing the potential effects of eucalypt plantations at several levels of study. Here we review the main conclusions of these investigations. Eucalypt plantations produced less litter than some deciduous forests. However, there were marked differences in timing of litterfall: litter production peaked during autumn in deciduous forests, whereas in the eucalypt forests it tended to peak in summer and to be more evenly distributed throughout the year. Despite these differences, the average standing stock of organic matter was higher in the eucalypt than in the deciduous forest. This may be attributed to (1) the occurrence of spates or heavy rain in autumn, the period of maximum litter fall in deciduous forests, and (2) bark accumulation in eucalypt forests. Because of differences in leaf composition, the nutrient input in eucalypt forests seems to be lower than in deciduous forests. The rate of decomposition of eucalypt leaves was strongly dependent on nutrients in the water: in nutrient-poor waters it was slower than that of most other leaf species, whereas in nutrient-rich waters it can be as fast as alder--a fast-decaying species. The biomass and cumulative diversity of aquatic hyphomycetes colonizing leaves did not differ between eucalypt and other native leaf species, but fungal sporulation generally peaked 2 weeks later on eucalypt leaves. This lag disappeared when lipids (but not polyphenolics) were chemically removed from eucalypt leaves. Similarly, addition of eucalypt oils to culture media retarded or suppressed fungal growth. Streams bordered by Eucalyptus had lower diversity of fungal spores (but similar spore densities) in Portugal; less consistent patterns were found in similar experiments in Spain. Eucalyptus leaves proved to be poor food for shredders. Under laboratory conditions leaves of Eucalyptus ranked low in food selection experiments using native shredders. The same shredders failed to grow and died when fed exclusively eucalypt leaves. The removal of oils from eucalypt leaves resulted in increased feeding rates, whereas the transfer of oils to alder leaves resulted in decreased feeding rates. The effect of eucalypt plantations on stream invertebrate communities is not very consistent. In nutrient-poor waters, fewer invertebrates colonized eucalypt than alder leaves, but this effect was mitigated after a microbial conditioning period in nutrient-rich waters. Portuguese streams bordered by Eucalyptus had lower numbers of invertebrates than streams surrounded by deciduous forests. In Spanish streams differences were less marked and nonexistent when looking at the composition of the communities, which change more from year to year than from site to site. Most of the eucalypt streams studied in Portugal and Spain dried up in summer, a fact that might reflect an increase in soil hydrophobity produced by Eucalyptus plantations. The very short planting-to-harvest period of eucalypt plantations results in additional impacts, such as soil loss, siltation of streams, or reduced amounts of woody debris in stream channels, which affects their capacity to retain leaf-litter, as well as the availability of habitat for invertebrates and fish. The studies by the Portuguese and Spanish research teams confirm the importance of maintaining riparian buffer strips to reduce human impact on streams and rivers.

Effects of essential oils from Eucalyptus globulus leaves on soil organisms involved in leaf degradation.

The replacement of native Portuguese forests by Eucalyptus globulus is often associated with deleterious effects on terrestrial and aquatic communities. Several studies have suggested that such a phenomenon is linked with the leaf essential oils released into the environment during the Eucalyptus leaf degradation process. However, to date, the way these compounds affect leaf degradation in terrestrial systems i.e. by direct toxic effects to soil invertebrates or indirectly by affecting food of soil fauna, is still unknown. In order to explore this question, the effect of essential oils extracted from E. globulus leaves on terrestrial systems was investigated. Fungal growth tests with species known as leaf colonizers (Mucor hiemalis, Alternaria alternata, Penicillium sp., Penicillium glabrum and Fusarium roseum) were performed to evaluate the antifungal effect of essential oils. In addition, a reproduction test with the collembolans Folsomia candida was done using a gradient of eucalyptus essential oils in artificial soil. The influence of essential oils on feeding behaviour of F. candida and the isopods Porcellio dilatatus was also investigated through food avoidance and consumption tests. Eucalyptus essential oils were lethal at concentrations between 2.5-20 µL/mL and inhibited growth of all fungal species between 1.25-5 µL/mL. The collembolan reproduction EC50 value was 35.0 (28.6-41.2) mg/kg and both collembola and isopods preferred leaves without oils. Results suggested that the effect of essential oils in leaf processing is related to direct toxic effects on fungi and soil fauna and to indirect effects on the quality and availability of food to soil invertebrates.