Sofia Alexandra Ferreira Duarte

Aquatic hyphomycete diversity and identity affect leaf litter decomposition in microcosms

We conducted a microcosm experiment with monocultures and all possible combinations of four aquatic hyphomycete species, Articulospora tetracladia, Flagellospora curta, Geniculospora grandis and Heliscus submersus, to examine the potential effects of species richness on three functional aspects: leaf litter decomposition (leaf mass loss), fungal production (ergosterol buildup) and reproductive effort (released spores). Both species richness and identity significantly affected fungal biomass and conidial production (number and biomass of released spores), whereas only species identity had a significant effect on leaf mass loss. In mixed cultures, all measures of fungal functions were greater than expected from the weighted performances of participating species in monoculture. Mixed cultures outperformed the most active monoculture for biomass accumulation but not for leaf mass loss and conidial production. The three examined aspects of aquatic hyphomycete activity tended to increase with species richness, and a complementary effect was unequivocally demonstrated for fungal biomass. Our results also suggest that specific traits of certain species may have a greater influence on ecosystem functioning than species number.

Microbial Decomposer Communities Are Mainly Structured by Trophic Status in Circumneutral and Alkaline Streams

ABSTRACT In streams, the release of nitrogen and phosphorus is reported to affect microbial communities and the ecological processes they govern. Moreover, the type of inorganic nitrogen (NO3, NO2, or NH4) may differently impact microbial communities. We aimed to identify the environmental factors that structure aquatic microbial communities and drive leaf litter decomposition along a gradient of eutrophication. We selected five circumneutral (Portuguese) and five alkaline (French) streams differing in nutrient concentrations to monitor mass loss of alder leaves, bacterial and fungal diversity by PCR-denaturing gradient gel electrophoresis, fungal biomass and reproduction, and bacterial biomass during 11 weeks of leaf immersion. The concentrations of inorganic nutrients in the stream water ranged from 5 to 300 μg liter−1 soluble reactive phosphorus, 0.30 to 5.50 mg liter−1 NO3-N, 2 to 103 μg liter−1 NO2-N, and <4 to 7,100 μg liter−1 NH4-N. Species richness was maximum in moderately anthropized (eutrophic) streams but decreased in the most anthropized (hypertrophic) streams. Different species assemblages were found in subsets of streams with different trophic statuses. In both geographic areas, the limiting nutrient, either nitrate or phosphate, stimulated the microbial activity in streams of intermediate trophic status. In the hypertrophic streams, fungal biomass and reproduction were significantly lower, and bacterial biomass dramatically decreased at the site with the highest ammonium concentration. The limiting nutrients that defined the trophic status were the main factor structuring fungal and bacterial communities, whatever the geographic area. A very high ammonium concentration in stream water most probably has negative impacts on microbial decomposer communities.

Effects of Zinc on Leaf Decomposition by Fungi in Streams: Studies in Microcosms

The effect of zinc on leaf decomposition by aquatic fungi was studied in microcosms. Alder leaf disks were precolonized for 15 days at the source of the Este River and exposed to different zinc concentrations during 25 days. Leaf mass loss, fungal biomass (based on ergosterol concentration), fungal production (rates of [1-14C]acetate incorporation into ergosterol), sporulation rates, and species richness of aquatic hyphomycetes were determined. At the source of the Este River decomposition of alder leaves was fast and 50% of the initial mass was lost in 25 days. A total of 18 aquatic hyphomycete species were recorded during 42 days of leaf immersion. Articulospora tetracladia was the dominant species, followed by Lunulospora curvula and two unidentified species with sigmoid conidia. Cluster analysis suggested that zinc concentration and exposure time affected the structure of aquatic hyphomycete assemblages, even though richness had not been severely affected. Both zinc concentration and exposure time significantly affected leaf mass loss, fungal production and sporulation, but not fungal biomass. Zinc exposure reduced leaf mass loss, inhibited fungal production and affected fungal reproduction by either stimulating or inhibiting sporulation rates. The results of this work suggested zinc pollution might depress leaf decomposition in streams due to changes in the structure and activity of aquatic fungi.

High Diversity of Fungi may Mitigate the Impact of Pollution on Plant Litter Decomposition in Streams

We investigated how a community of microbial decomposers adapted to a reference site responds to a sudden decrease in the water quality. For that, we assessed the activity and diversity of fungi and bacteria on decomposing leaves that were transplanted from a reference (E1) to a polluted site (E2), and results were compared to those from decomposing leaves either at E1 or E2. The two sites had contrasting concentrations of organic and inorganic nutrients and heavy metals in the stream water. At E2, leaf decomposition rates, fungal biomass, and sporulation were reduced, while bacterial biomass was stimulated. Fungal diversity was four times lower at the polluted site. The structure of fungal community on leaves decomposing at E2 significantly differed from that decomposing at E1, as indicated by the principal response curves analysis. Articulospora tetracladia, Anguillospora filiformis, and Lunulospora curvula were dominant species on leaves decomposing at E1 and were the most negatively affected by the transfer to the polluted site. The transfer of leaves colonized at the reference site to the polluted site reduced fungal diversity and sporulation but not fungal biomass and leaf decomposition. Overall, results suggest that the high diversity on leaves from the upstream site might have mitigated the impact of anthropogenic stress on microbial decomposition of leaves transplanted to the polluted site.

Mixtures of zinc and phosphate affect leaf litter decomposition by aquatic fungi in streams

To better understand the impacts of multiple stressors in freshwaters, we investigated the effects of mixtures of zinc and inorganic phosphorus on microbial decomposition of leaf litter. Alder leaves were colonized in a stream and placed in microcosms with stream water supplemented or not with 3 concentrations of zinc (Zn up to 9.8 mg/l) or phosphate (P-PO(4)(3-) up to 0.5 mg/l), alone and in all possible combinations. We measured leaf mass loss, and fungal biomass, reproduction and diversity. In control microcosms, 23 species of aquatic hyphomycetes were identified on leaves, and the exposure to the highest zinc concentration reduced diversity to 14 species. Articulosporatetracladia was the dominant species followed by Flagellospora sp. and Alatosporaacuminata. The exposure to phosphate increased the contribution of A.acuminata, but this species was negatively affected by zinc. Under high zinc stress, Varicosporiumelodeae increased its contribution to the total conidial production. The exposure to high zinc concentration, alone or in mixtures with phosphate, led to shifts in fungal community structure, as indicated by cluster analysis based on sporulation data and denaturing gradient gel electrophoresis (DGGE) fingerprints of fungal DNA. These changes were accompanied by a reduction in leaf decomposition, particularly in mixtures with high Zn concentration, in which leaf mass loss was 30% lower than in the control. This suggests that the co-occurrence of zinc and phosphate may have negative effects on stream ecosystem functioning. However, we did not detect decreased leaf-associated fungal biomass and sporulation, probably because a delay in fungal colonization occurred due to the presence of stressors.

Denaturing Gradient Gel Electrophoresis (DGGE) in microbial ecology : insights from freshwaters

The Portuguese Foundation for the Science and Technology supported this work (PTDC/AAC-AMB/113746/2009) and S. Duarte (SFRH/BPD/47574/2008).

Intraspecific variation of the aquatic fungus Articulospora tetracladia: an ubiquitous perspective.

The worldwide-distributed aquatic fungus Articulospora tetracladia Ingold is a dominant sporulating species in streams of the Northwest Iberian Peninsula. To elucidate the genetic diversity of A. tetracladia, we analyzed isolates collected from various types of plant litter or foam in streams from North and Central Portugal and North Spain, between 2000 and 2010. Genetic diversity of these fungal populations was assessed by denaturing gradient gel electrophoresis (DGGE) fingerprints and by using ITS1-5.8S-ITS2 barcodes. Moreover, ITS1-5.8S-ITS2 barcodes of A. tetracladia reported in other parts of the world (Central Europe, United Kingdom, Canada, Japan and Malaysia) were retrieved from the National Center for Biotechnology (NCBI) and the National Institute of Technology and Evaluation Biological Resource Center (NBRC) to probe into genetic diversity of A. tetracladia. PCR-DGGE of ITS2 region of 50 Iberian fungal isolates distinguished eight operational taxonomic units (OTUs), which were similar to those obtained from neighboring trees based on ITS2 gene sequences. On the other hand, ITS1-5.8S-ITS2 barcodes of 68 fungal isolates yielded nine OTUs, but five fungal isolates were not assigned to any of these OTUs. Molecular diversity was highest for OTU-8, which included only European isolates. Two haplotypes were observed within OTU-8 and OTU-9, while only one haplotype was found within each of the remaining OTUs. Malaysia did not share haplotypes with other countries. Overall results indicate that, apart from the Malaysian genotypes, A. tetracladia genotypes were geographically widespread irrespective of sampling time, sites or substrates. Furthermore, PCR-DGGE appeared to be a rapid tool for assessing intraspecific diversity of aquatic hyphomycetes.

Differences in the sensitivity of fungi and bacteria to season and invertebrates affect leaf litter decomposition in a Mediterranean stream

Microorganisms are key drivers of leaf litter decomposition; however, the mechanisms underlying the dynamics of different microbial groups are poorly understood. We investigated the effects of seasonal variation and invertebrates on fungal and bacterial dynamics, and on leaf litter decomposition. We followed the decomposition of Populus nigra litter in a Mediterranean stream through an annual cycle, using fine and coarse mesh bags. Irrespective of the season, microbial decomposition followed two stages. Initially, bacterial contribution to total microbial biomass was higher compared to later stages, and it was related to disaccharide and lignin degradation; in a later stage, bacteria were less important and were associated with hemicellulose and cellulose degradation, while fungi were related to lignin decomposition. The relevance of microbial groups in decomposition differed among seasons: fungi were more important in spring, whereas in summer, water quality changes seemed to favour bacteria and slowed down lignin and hemicellulose degradation. Invertebrates influenced litter-associated microbial assemblages (especially bacteria), stimulated enzyme efficiencies and reduced fungal biomass. We conclude that bacterial and fungal assemblages play distinctive roles in microbial decomposition and differ in their sensitivity to environmental changes, ultimately affecting litter decomposition, which might be particularly relevant in highly seasonal ecosystems, such as intermittent streams.

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.

The role of the freshwater shrimp atyaephyra desmarestii in leaf litter breakdown in streams

In aquatic ecosystems, microorganisms and invertebrates provide critical links between plant detritus and higher trophic levels. Atyaephyra desmarestii is an omnivorous decapod that inhabits freshwaters and exhibits high tolerance to temperature oscillations and high ability to colonize new habitats. Although A. desmarestii is able to ingest a variety of foods, few studies have been conducted to elucidate the role of this freshwater shrimp on detritus breakdown in streams. In this study, A. desmarestii was allowed to feed on conditioned or unconditioned alder and eucalyptus leaves in microcosms with or without access to its fecal pellets. At the end of the experiment, total body length of the animals was measured, and the remaining leaves and fecal pellets were used for dry mass quantification and assessment of bacterial and fungal diversity by denaturing gradient gel electrophoresis (DGGE). Cluster analyses of DGGE fingerprints indicated that the major differences in microbial communities on leaves were between leaf types, while on fecal pellets were between conditioned and unconditioned leaves. However, the consumption rate by the shrimp did not differ between leaf types, and was significantly higher on leaves conditioned by microorganisms and in treatments without access to feces. In treatments without access to feces, the production of feces and fine particulate organic matter was also significantly higher for conditioned leaves. Overall, our results support the feeding plasticity of A. desmarestii and its potential role in plant litter breakdown in streams. This might have implications for maintaining stream ecosystem functioning, particularly if more vulnerable shredders decline.