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.

Fungal propagules and DNA in feces of two detritus-feeding amphipods.

Aquatic shredders (leaf-eating invertebrates) preferentially ingest and digest leaves colonized by aquatic hyphomycetes (fungi). This activity destroys leaf-associated fungal biomass and detritial resources in streams. Fungal counter-adaptations may include the ability to survive passage through the invertebrate's digestive tract. When fecal pellets of Gammarus tigrinus and Hyalella azteca were incubated with sterile leaves, spores of nine (G. tigrinus) and seven (H. azteca) aquatic hyphomycete species were subsequently released from the leaves, indicating the presence of viable fungal structures in the feces. Extraction, amplification, and sequencing of DNA from feces revealed numerous fungal phylotypes, two of which could be assigned unequivocally to an aquatic hyphomycete. The estimated contributions of major fungal groups varied depending on whether 18S or ITS sequences were amplified and cloned. We conclude that a variable proportion of fungal DNA in the feces of detritivores may originate from aquatic hyphomycetes. Amplified DNA may be associated with metabolically active, dormant, or dead fungal cells.

Realized fungal diversity increases functional stability of leaf litter decomposition under zinc stress.

Freshwaters include some of the most impaired systems on Earth with high rates of species loss, underscoring the significance of investigating whether ecosystems with fewer species will be able to maintain ecological processes. The environmental context is expected to modulate the effects of declining diversity. We conducted microcosm experiments manipulating fungal inoculum diversity and zinc concentration to test the hypothesis that fungal diversity determines the susceptibility of leaf litter decomposition to Zn stress. Realized fungal diversity was estimated by counting released spores and by measuring species-specific biomasses via denaturing gradient gel electrophoresis. In the absence of Zn, positive diversity effects were found for leaf mass loss and fungal biomass through complementary interactions and due to the presence of key species. The variability of leaf decomposition decreased with increasing species number (portfolio effect), particularly under Zn stress. Results suggest that the effect of species loss on ecosystem stability may be exacerbated at higher stress levels.

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.

Rapid characterization of aquatic hyphomycetes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Protein fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI--TOF MS) is a rapid, reliable, and economical method to characterize isolates of terrestrial fungi and other microorganisms. The objective of our study was to evaluate the suitability of MALDI-TOF MS for the identification of aquatic hyphomycetes, a polyphyletic group of fungi that play crucial roles in stream ecosystems. To this end, we used 34 isolates of 21 aquatic hyphomycete species whose identity was confirmed by spore morphology and internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) nuc rDNA sequencing. We tested the efficiency of three protein extraction methods, including chemical and mechanical treatments using 13 different protocols, with the objective of producing high-quality MALDI-TOF mass spectra. In addition to extraction protocols, mycelium age was identified as a key parameter affecting protein extraction efficiency. The dendrogram based on mass-spectrum similarity indicated good and relevant taxonomic discrimination; the tree structure was comparable to that of the phylogram based on ITS sequences. Consequently, MALDI-TOF MS could reliably identify the isolates studied and provided greater taxonomic accuracy than classical morphological methods. MALDI-TOF MS seems suited for rapid characterization and identification of aquatic hyphomycete species.

Biodiversity of leaf litter fungi in streams along a latitudinal gradient.

Global patterns of biodiversity have emerged for soil microorganisms, plants and animals, and the extraordinary significance of microbial functions in ecosystems is also well established. Virtually unknown, however, are large-scale patterns of microbial diversity in freshwaters, although these aquatic ecosystems are hotspots of biodiversity and biogeochemical processes. Here we report on the first large-scale study of biodiversity of leaf-litter fungi in streams along a latitudinal gradient unravelled by Illumina sequencing. The study is based on fungal communities colonizing standardized plant litter in 19 globally distributed stream locations between 69°N and 44°S. Fungal richness suggests a hump-shaped distribution along the latitudinal gradient. Strikingly, community composition of fungi was more clearly related to thermal preferences than to biogeography. Our results suggest that identifying differences in key environmental drivers, such as temperature, among taxa and ecosystem types is critical to unravel the global patterns of aquatic fungal diversity.

Fungi in a heavy metal precipitating stream in the Mansfeld mining district, Germany.

Fungal growth on alder leaves was studied in two heavy metal polluted streams in central Germany. The aim of the study was to examine previously observed differences in leaf decomposition rates, heavy metal precipitation and fungal involvement in these processes at the microscopic level. Ergosterol analyses indicated that neither habitat was optimal for fungi, but leaves exposed at the less polluted site (H8) decomposed rapidly and were colonized externally and internally by fungi and other microorganisms. Leaves exposed at the more polluted site (H4) decomposed very slowly and fungal colonization was restricted to external surfaces. An amorphous organic layer, deposited within 24 h of exposure, quickly became covered with a pale blue-green crystalline deposit (zincowoodwardite) with significant amounts of Al, S, Cu and Zn, determined by energy dispersive X-ray spectroscopy (EDS). Scanning electron microscopy (SEM) analysis of the precipitate revealed a branching arrangement of the precipitated particles caused by the presence of fungal hyphae growing on the surface. Hyphae that were not disturbed by handling were usually completely encased in the precipitate, but hyphae did not contain EDS-detectable amounts of precipitate metals. Elemental analysis using inductively coupled plasma (ICP) atomic emission spectrometry and ICP mass spectrometry revealed continuing accumulation of Zn, Cu and several other metals/metalloids on and in leaves. The formation of metal precipitates on various artificial substrates at site H4 was much reduced compared to leaves, which we attribute to the absence of fungal colonization on the artificial substrates. We could not determine whether fungi accelerate the precipitation of heavy metals at site H4, but mycelial growth on leaves continues to create new surfaces and therefore thicker layers of precipitate on leaves compared to artificial substrates.

Articulospora - Phylogeny vs morphology.

The taxonomy of the aquatic hyphomycete genus Articulospora (Ascomycota, Pezizomycotima, Leotiales, Helotiaceae) is based on the morphology of the generative phase of its lifecycle. The type species is Articulospora tetracladia, which is distributed worldwide. Its most frequent populations in nature have dimorphic conidia, differing by the extent of conidial branching (i.e., one or two levels of branching). Some strains, stable in culture, produce exclusively conidia of one type. With the molecular analyses employed here and the relatively low number of available isolates (20), separation based on branching of conidia has not been fully supported. Therefore we propose to retain the broad concept of A. tetracladia with dimorphic conidia. Among the three gene sequences tested as potential barcodes, the internal transcribed spacer (ITS) gene was the most promising region. All strains yielded amplifiable DNA which provided adequate resolution, according to accepted ranges in inter/intraspecific differences, to differentiate among the three Articulospora and two Fontanospora species that were tested (Articulospora atra, Articulospora proliferata, A. tetracladia, Fontanospora eccentrica, Fontanospora fusiramosa). D1/D2 primers also permitted amplification in all strains, however without much resolution. Amplification of the COX1 gene sequence was least consistent.

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'.

Taxa-area relationship of aquatic fungi on deciduous leaves.

One of the fundamental patterns in macroecology is the increase in the number of observed taxa with size of sampled area. For microbes, the shape of this relationship remains less clear. The current study assessed the diversity of aquatic fungi, by the traditional approach based on conidial morphology (captures reproducing aquatic hyphomycetes) and next generation sequencing (NGS; captures other fungi as well), on graded sizes of alder leaves (0.6 to 13.6 cm2). Leaves were submerged in two streams in geographically distant locations: the Oliveira Stream in Portugal and the Boss Brook in Canada. Decay rates of alder leaves and fungal sporulation rates did not differ between streams. Fungal biomass was higher in Boss Brook than in Oliveira Stream, and in both streams almost 100% of the reads belonged to active fungal taxa. In general, larger leaf areas tended to harbour more fungi, but these findings were not consistent between techniques. Morphospecies-based diversity increased with leaf area in Boss Brook, but not in Oliveira Stream; metabarcoding data showed an opposite trend. The higher resolution of metabarcoding resulted in steeper taxa-accumulation curves than morphospecies-based assessments (fungal conidia morphology). Fungal communities assessed by metabarcoding were spatially structured by leaf area in both streams. Metabarcoding promises greater resolution to assess biodiversity patterns in aquatic fungi and may be more accurate for assessing taxa-area relationships and local to global diversity ratios.

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.

Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species - A proof-of-concept using Alatospora pulchella.

Traditional methods to identify aquatic hyphomycetes rely on the morphology of released conidia, which can lead to misidentifications or underestimates of species richness due to convergent morphological evolution and the presence of non-sporulating mycelia. Molecular methods allow fungal identification irrespective of the presence of conidia or their morphology. As a proof-of-concept, we established a quantitative real-time polymerase chain reaction (qPCR) assay to accurately quantify the amount of DNA as a proxy for the biomass of an aquatic hyphomycete species (Alatospora pulchella). Our study showed discrimination even among genetically closely-related species, with a high sensitivity and a reliable quantification down to 9.9 fg DNA (3 PCR forming units; LoD) and 155.0 fg DNA (47 PCR forming units; LoQ), respectively. The assay's specificity was validated for environmental samples that harboured diverse microbial communities and likely contained PCR-inhibiting substances. This makes qPCR a promising tool to gain deeper insights into the ecological roles of aquatic hyphomycetes and other microorganisms.

Metabarcoding-based fungal diversity on coarse and fine particulate organic matter in a first-order stream in Nova Scotia, Canada.

Most streams receive substantial inputs of allochthonous organic material in the form of leaves and twigs (CPOM , coarse particulate organic matter). Mechanical and biological processing converts this into fine particulate organic matter (FPOM). Other sources of particles include flocculated dissolved matter and soil particles. Fungi are known to play a role in the CPOM conversion process, but the taxonomic affiliations of these fungi remain poorly studied. The present study seeks to shed light on the composition of fungal communities on FPOM and CPOM as assessed in a natural stream in Nova Scotia, Canada. Maple leaves were exposed in a stream for four weeks and their fungal community evaluated through pyrosequencing. Over the same period, four FPOM size fractions were collected by filtration and assessed. Particles had much lower ergosterol contents than leaves, suggesting major differences in the extent of fungal colonization. Pyrosequencing documented a total of 821 fungal operational taxonomic units (OTU), of which 726 were exclusive to particles and 47 to leaf samples. Most fungal phyla were represented, including yeast lineages (e.g., Taphrinaceae and Saccharomycotina), Basidiomycota, Chytridiomycota and Cryptomycota, but several classes of Pezizomycontina (Ascomycota) dominated. Cluster dendrograms clearly separated fungal communities from leaves and from particles. Characterizing fungal communities may shed some light on the processing pathways of fine particles in streams and broadens our view of the phylogenetic composition of fungi in freshwater ecosystems.

The contribution of fungal enzymes to the digestion of leaves by Gammarus fossarum Koch (Amphipoda).

Gut extracts of Gammarus fossarum liberated reducing substances (at pH values ≤7) and amino acids (pH≥7) from freshly shed oak leaves only after removal of soluble leaf phenols. When carboxymethylcellulose was used at a concentration equal to that of leaf cellulose, release of reducing substances was considerably higher. Fungal enzymes extracted from decomposing leaves were active against structural carbohydrates but showed no proteolytic activity. At low pH values, they retained their full activity in the presence of gut enzymes of G. fossarum, at higher pH values they were inhibited. Conditioned leaves released larger amounts of reducing substances and amino acids when exposed to gut enzymes. The improvement varies with the fungal species used for conditioning and with the length of the conditioning period. The digestibility of leaf carbohydrates and proteins reached separate peaks and then declined. Fungal carbohydrases ingested by G. fossarum retained some activity for up to 4h.

Leaf-eating invertebrates as competitors of aquatic hyphomycetes.

Leaf-eating invertebrates selectively ingest leaf areas rich in fungal cells. The effect of this process on coincident and cumulative species diversity (species numbers and evenness) of the fungi was studied on 3 substrates (oak leaves, larch and spruce needles) in 2 hardwater and 2 softwater streams. Cumulative species number of colonizing fungi follows the equation S=k·A z(A=area below decay curve of the substrate, k=substrate-specific constant, Z=0.47). Higher feeding activity means faster weight loss of the substrate which leads to lower species richness of the fungi. The opposite is true for early successional stages on larch needles. Evenness of the fungi (distribution of individuals among species) is negatively correlated with feeding intensity by invertebrates, as measured by increased decay rates. The overall effect of leaf-eating invertebrates on aquatic hyphomycetes resembles that of potent competitors preempting substrate otherwise used by a late successional tail of relatively rare fungi.

Leaf-conditioning by microorganisms.

Many detritus-feeders prefer dead leaves which are colonized by microorganisms-i.e. conditioned leaves, over freshly fallen or sterile leaves. Traditionally, this has been attributed to the build-up of microbial cells on the substrate. Two experiments show that changes in the leaf itself, brought about by microbial excretions and secretions, or by a hydrolytic agent (hot HCl), can also increase its palatability to the detritus-feeding amphipod Gammarus pseudolimnaeus.

Pellet size affects mycelial ergosterol content in aquatic hyphomycetes.

Ergosterol was measured in mycelia of seven species of aquatic hyphomycetes grown in malt-extract broth. The harvested 21 d old pellets were grouped into 5-6 classes based on size, which were analyzed separately. In all but one species, there was a significant, positive correlation between the amount of ergosterol per unit mass and pellet diameter. Ignoring this correlation could result in the misleading conclusion that there is no relationship between mycelial mass and its absolute ergosterol content. The highest ergosterol concentrations were close to the average generally used to convert the amount of ergosterol in environmental samples to fungal biomass; the average was about half that value.

Preliminary insights into the phylogeography of six aquatic hyphomycete species.

Aquatic hyphomycetes occur worldwide on a wide range of plant substrates decomposing in freshwaters, and are known to play a key role in organic matter turnover. The presumed worldwide distribution of many aquatic hyphomycete species has been based on morphology-based taxonomy and identification, which may overlook cryptic species, and mask global-scale biogeographical patterns. This might be circumvented by using DNA sequence data. The internal transcribed spacer (ITS) region from rDNA was recently designated as the most suitable barcode for fungal identification. In this study, we generated ITS barcodes of 130 isolates belonging to 6 aquatic hyphomycete species (Anguillospora filiformis, Flagellospora penicillioides, Geniculospora grandis, Lunulospora curvula, Tetrachaetum elegans and Tricladium chaetocladium), and collected from streams of Southwest Europe (86 isolates) and East Australia (44 isolates). European and Australian populations of 4 species (A. filiformis, F. penicillioides, G. grandis and T. elegans) grouped into different clades, and molecular diversity indices supported significant differentiation. Continents did not share haplotypes, except for T. chaetocladium. Overall results show substantial population diversity for all tested species and suggests that the biogeography of aquatic hyphomycetes may be species-specific.

Quantitative methods for the analysis of zoosporic fungi.

Quantitative estimations of zoosporic fungi in the environment have historically received little attention, primarily due to methodological challenges and their complex life cycles. Conventional methods for quantitative analysis of zoosporic fungi to date have mainly relied on direct observation and baiting techniques, with subsequent fungal identification in the laboratory using morphological characteristics. Although these methods are still fundamentally useful, there has been an increasing preference for quantitative microscopic methods based on staining with fluorescent dyes, as well as the use of hybridization probes. More recently however PCR based methods for profiling and quantification (semi- and absolute) have proven to be rapid and accurate diagnostic tools for assessing zoosporic fungal assemblages in environmental samples. Further application of next generation sequencing technologies will however not only advance our quantitative understanding of zoosporic fungal ecology, but also their function through the analysis of their genomes and gene expression as resources and databases expand in the future. Nevertheless, it is still necessary to complement these molecular-based approaches with cultivation-based methods in order to gain a fuller quantitative understanding of the ecological and physiological roles of zoosporic fungi.

Reproduction of aquatic hyphomycetes at low concentrations of Ca2+, Zn2+, Cu2+, and Cd2+.

Maple leaf disks were conditioned in a stream for three weeks and then aerated for 2 d in distilled water to induce fungal sporulation. The release of aquatic hyphomycete spores increased when the water was supplemented with low concentrations of Ca(2+) (5 µg/L), Zn(2+) (2.5 µg/L), Cu(2+) (0.5 µg/L), or Cd(2+) (0.125 µg/L). Higher supplement concentrations inhibited sporulation. Over the concentration range used, the sporulation response was generally best described by a quadratic regression, suggesting a biphasic or hormetic response. A similar pattern was found with the number of fungal species as the dependent variable. Anguillospora filiformis and Anguillospora longissima were generally least inhibited by metal supplements, and Ca(2+) was the least and Cd(2+) the most toxic metal. Combinations of metals had a more severe effect on fungal sporulation than predicted from addition of the effects of the metals in isolation. The biological significance of the hormetic response is unclear; however, acknowledging it is clearly relevant for establishing guidelines or recommendations in toxicology.