Population structure of Guyanagaster necrorhizus supports termite dispersal for this enigmatic fungus

Understanding how species accomplish dispersal of their propagules can shed light on how they are adapted for their ecosystem. Guyanagaster necrorhizus is a sequestrate fungus, meaning its dispersal propagules, or spores, are entirely enclosed within a fruiting body, termed a sporocarp. This fungus is most closely related to Armillaria and its allies. While Armillaria species form mushrooms and have forcibly discharged spores, G. necrorhizus spores have lost this ability, and by necessity, must be passively dispersed. However, G. necrorhizus does not possess characteristics of other sequestrate fungi with known dispersal mechanisms. Repeated observations of termites feeding on G. necrorhizus sporocarps, and spores subsequently adhering to their exoskeletons, led to the hypothesis that termites disperse G. necrorhizus spores. To test this hypothesis, we used microsatellite markers and population genetics analyses to understand patterns of clonality and population structure of G. necrorhizus. While Armillaria individuals can spread vegetatively over large areas, high genotypic diversity in G. necrorhizus populations suggests spores are the primary mode of dispersal. Spatial genetic structure analyses show that G. necrorhizus sporocarps within 238 m of each other are more closely related than would be expected by chance and conservative estimates from population assignment tests suggest gene flow no longer occurs between sporocarps separated by 2 km. These distances are consistent with previous studies analysing foraging distances of the termites found associated with G. necrorhizus sporocarps. Termites have rarely been recorded to specifically target fungal sporocarps, making this a potentially novel fungal–insect interaction.     

Beetles provide directed dispersal of viable spores of a keystone wood decay fungus

Wood decay fungi are considered to be dispersed by wind, but dispersal by animals may also be important, and more so in managed forests where dead wood is scarce. We investigated whether beetles could disperse spores of the keystone species Fomitopsis pinicola. Beetles were collected on sporocarps and newly felled spruce logs, a favourable habitat for spore deposition. Viable spores (and successful germination) of F. pinicola were detected by dikaryotization of monokaryotic bait mycelium from beetle samples. Viable spores were on the exoskeleton and in the faeces of all beetles collected from sporulating sporocarps. On fresh spruce logs, nine beetle species transported viable spores, of which several bore into the bark. Our results demonstrate that beetles can provide directed dispersal of wood decay fungi. Potentially, it could contribute to a higher persistence of some species in fragmented forests where spore deposition by wind on dead wood is less likely.     

Records of hypogeous mycorrhizal fungi in the diet of some Western Australian bolboceratine beetles (Coleoptera: Geotrupidae, Bolboceratinae)

In order to assess the possible contribution of bolboceratine beetles to the dispersal of mycorrhizal fungal spores, faeces and/or gut contents of adults of several species and genera obtained from burrows or collected at lights at night were examined microscopically. Two species of Blackbolbus ( frontalis and fucinus ) were found to have fed on different species of hypogeous sporocarps (truffles). Furthermore, adults of Bbo. frontalis were found in burrows with truffles of the genera Amarrendia , Hysterangium and Scleroderma . Specimens of some Blackburnium , Bolboleaus and Bolborhachium species, on the other hand, were found to have ingested diffuse glomeralean mycelium and spores along with varying quantities of soil. Limited evidence of broad-scale distribution of spores was obtained. Some Scleroderma truffles found in burrows of Bbo. frontalis were honeycombed and inhabited by numerous nitidulid beetles identified as Thalycrodes mixta . Two truffles identified as Hysterangium found in soil close to a burrow of Bbo. frontalis were infested with nematodes.     

Infiltration by alien predators into invertebrate food webs in Hawaii: a molecular approach

Abstract Alien invertebrate predators have been introduced to Hawaii to control pests, particularly in lowland areas where most crops are grown. We developed techniques for assessing the impact of these predators on native food webs in relatively pristine upland areas where, it was hypothesized, few lowland predators might be found. Predator densities were assessed along transects within the Alakaii Swamp on Kaua'i. The most numerous alien biocontrol agents found were Halmus chalybeus (Coccinellidae), a species known to feed on Lepidoptera eggs. Laboratory experiments were conducted using two genera of endemic Lepidoptera, Scotorythra and Eupithecia (Geometridae), that are of considerable conservation value, the former because of its recent speciation across Hawaii, the latter for its unique predatory larvae. Techniques were developed for detecting Lepidoptera DNA within the guts of alien predators using prey-specific PCR primers. General primers amplified fragments of the mitochondrial cytochrome oxidase I gene from beetles and Lepidoptera. The sequences were aligned and used successfully to design target-specific primers for general detection of the remains of Geometridae and for particular species, including Scotorythra rara and Eupithecia monticolans. DNA fragments amplified were short [140-170 base pairs (bp)], optimizing detection periods following prey ingestion. Trials using the introduced biocontrol agent Curinus coeruleus (Coccinellidae) demonstrated detection of Lepidoptera DNA fragments = 151 bp in 85-100% of beetles after 24 h digestion of an early instar larva. This study provides a framework for future use of molecular gut analysis in arthropod conservation ecology and food web research with considerable potential for quantifying threats to endemic species in Hawaii and elsewhere.     

Resolving the predator first paradox: Arthropod predator food webs in pioneer sites of glacier forelands

Primary succession on bare ground surrounded by intact ecosystems is, during its first stages, characterized by predator‐dominated arthropod communities. However, little is known on what prey sustains these predators at the start of succession and which factors drive the structure of these food webs. As prey availability can be extremely patchy and episodic in pioneer stages, trophic networks might be highly variable. Moreover, the importance of allochthonous versus autochthonous food sources for these pioneer predators is mostly unknown. To answer these questions, the gut content of 1,832 arthropod predators, including four species of carabid beetles, two lycosid and several linyphiid spider species caught in early and late pioneer stages of three glacier forelands, was screened molecularly to track intraguild and extraguild trophic interactions among all major prey groups occurring in these systems. Two‐thirds of the 2,310 identified food detections were collembolans and intraguild prey, while one‐third were allochthonous flying insects. Predator identity and not successional stage or valley had by far the strongest impact on the trophic interaction patterns. Still, the variability of prey spectra increased significantly from early to late pioneer stage, as did the niche width of the predators. As such the structure of pioneer arthropod food webs in recently deglaciated Alpine habitats seems to be driven foremost by predator identity while site and early successional effects contribute to a lesser extent to food web variability. Our findings also suggest that in these pioneer sites, predatory arthropods depend less on allochthonous aeolian prey but are mainly sustained by prey of local production.     

Genet dynamics and ecological functions of the pioneer ectomycorrhizal fungi Laccaria amethystina and Laccaria laccata in a volcanic desert on Mount Fuji

To understand the reproduction of the pioneer ectomycorrhizal fungi Laccaria amethystina and Laccaria laccata in a volcanic desert on Mount Fuji, Japan, the in situ genet dynamics of sporocarps were analysed. Sporocarps of the two Laccaria species were sampled at fine and large scales for 3 and 2 consecutive years, respectively, and were genotyped using microsatellite markers. In the fine-scale analysis, we found many small genets, the majority of which appeared and disappeared annually. The high densities and annual renewal of Laccaria genets indicate frequent turnover by sexual reproduction via spores. In the large-scale analysis, we found positive spatial autocorrelations in the shortest distance class. An allele-clustering analysis also showed that several alleles were distributed in only a small, localised region. These results indicate that Laccaria spores contributing to sexual reproduction may be dispersed only short distances from sporocarps that would have themselves been established via rare, long-distance spore dispersal. This combination of rare, long-distance and frequent, short-distance Laccaria spore dispersal is reflected in the establishment pattern of seeds of their host, Salix reinii.     

Wood-inhabiting insects can function as targeted vectors for decomposer fungi

Most wood-inhabiting fungi are assumed to be dispersed primarily by wind, with the exception of a few species involved in mutualistic relationships with insects. In this study we tested whether several species of wood-inhabiting insects can function as dispersal vectors for non-mutualistic fungi, which would indicate that wood-inhabiting fungi can benefit from targeted animal-mediated dispersal. We sampled wood-inhabiting beetles (Coleoptera) from freshly felled wood experimentally added to forests and used DNA metabarcoding to investigate the fungal DNA carried by these insects. Staphylinid beetles rarely contained fungal DNA, while Endomychus coccineus, Glischrochilus hortensis and Glischrochilus quadripunctatus frequently carried fungal DNA with a composition specific to the insect taxon. A large proportion of the obtained fungal sequences (34%) represented decomposer fungi, including well-known wood-decay fungi such as Fomitopsis pinicola, Fomes fomentarius, Trichaptum abietinum and Trametes versicolor. Scanning electron microscopy further showed that some of the fungal material was carried as spores or yeast cells on the insect exoskeletons. Our results suggest that insect-vectored dispersal is of broader importance to wood-inhabiting fungi than previously assumed.     

Soil burrowing Muscina angustifrons (Diptera: Muscidae) larvae excrete spores capable of forming mycorrhizae underground

Muscina angustifrons (Diptera: Muscidae) is a mycophagous species that exploits a variety of fungi, including ectomycorrhizal fungi. Larvae of this species have been shown to feed on sporocarps (including spores), and full-grown larvae leave sporocarps and pupate 0–6?cm below the soil surface. In this study, we examined whether M. angustifrons larvae are capable of transporting ectomycorrhizal fungal spores and enhancing ectomycorrhiza growth on host-plant roots. Full-grown larvae were found to move horizontally 10–20?cm from their feeding sites and burrow underground. These wandering larvae retained ectomycorrhizal fungal spores in their intestines, which were excreted following relocation to underground pupation sites. Excreted spores retained germination and infection capacities to form ectomycorrhiza on host-plant roots. In the infection experiments, ectomycorrhizal fungal spores applied in the vicinity of underground host-plant roots were more effective in forming ectomycorrhiza than those applied to the ground surface, suggesting that belowground transportation of spores by M. angustifrons larvae could enhance ectomycorrhizal formation. These results suggested that M. angustifrons larvae act as a short-distance spore transporter of ectomycorrhizal fungi.     

Diversity and abundance of resupinate thelephoroid fungi as ectomycorrhizal symbionts in Swedish boreal forests

Resupinate thelephoroid fungi (hereafter called tomentelloid fungi) have a world-wide distribution and comprise approximately 70 basidiomycete species with inconspicuous, resupinate sporocarps. It is only recently that their ability to form ectomycorrhizas (EM) has been realized, so their distribution, abundance and significance as mycobionts in forest ecosystems is still largely unexplored. In order to provide baseline data for future ecological studies of tomentelloid fungi, we explored their presence and abundance in nine Swedish boreal forests in which the EM communities had been analysed. Phylogenetic analyses were used to compare the internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) sequence data obtained from mycobionts on single ectomycorrhizal tips with that obtained from sporocarps of identified tomentelloid fungi. Five species of Tomentella and one species of Pseudotomentella were identified as ectomycorrhizal fungi. The symbiotic nature of Tomentella bryophila, T. stuposa, T. badia and T. atramentaria is demonstrated for the first time. T. stuposa and Pseudotomentella tristis were the most commonly encountered tomentelloid fungi, with the other species, including T. sublilacina, only being recorded from single stands. Overall, tomentelloid fungi were found in five of the studies, colonizing between 1 and 8% of the mycorrhizal root tips. Two of the five sites supported several tomentelloid species. Tomentelloid fungi appear to be relatively common ectomycorrhizal symbionts with a wide distribution in Swedish coniferous forests. The results are in accordance with accumulating data that fungal species which lack conspicuous sporocarps may be of considerable importance in EM communities.     

Fern and bryophyte endozoochory by slugs

Endozoochory plays a prominent role for the dispersal of seed plants, and dispersal vectors are well known. However, for taxa such as ferns and bryophytes, endozoochory has only been suggested anecdotally but never tested in controlled experiments. We fed fertile leaflets of three ferns and capsules of four bryophyte species to three slug species. We found that, overall, spores germinated from slug feces in 57.3 % of all 89 fern and in 51.3 % of all 117 bryophyte samples, showing that the spores survived gut passage of slugs. Moreover, the number of samples within which spores successfully germinated did not differ among plant species but varied strongly among slug species. This opens new ecological perspectives suggesting that fern and bryophyte endozoochory by gastropods is a so-far-overlooked mode of dispersal, which might increase local population sizes of these taxa by spore deposition on suitable substrates.     

A sensitive method for examining whole-cell biochemical composition in single cells of filamentous fungi using synchrotron FTIR spectromicroscopy

Cell function is related to cell composition. The asexual state of filamentous fungi (molds and mildews) has two main life cycle stages: vegetative hyphae for substrate colonization and nutrient acquisition, and asexual spores for survival and dispersal. Hyphal composition changes over a few tens of microns during growth and maturation; spores are different from hyphae. Most biochemical analyses are restricted to studying a few components at high spatial resolution (e.g. histochemistry) or many compounds at low spatial resolution (e.g. GC-MS). Synchrotron FTIR spectromicroscopy can be used to study fungal cell biology by fingerprinting varieties of carbohydrates, proteins, and lipids at about 6 microm spatial resolution. FTIR can distinguish fungal species and changes during hyphal growth, and reveals that even fungi grown under optimal vs mildly stressed conditions exhibit dramatic biochemical changes without obvious morphological effects. Here we compare hypha and spore composition of two fungi, Neurospora and Rhizopus. There are clear biochemical changes when Neurospora hyphae commit to spore development, during spore maturation and following germination, many of which are consistent with results from molecular genetics, but have not been shown before at high spatial resolution. Rhizopus spores develop within a fluid-containing sporangium that becomes dry at maturity. Rhizopus spores had similar protein content and significantly more carbohydrate than the sporangial fluid, both of which are novel findings.     

The impact of arthropods on fungal community structure in Lascaux Cave

Aims:  To determine the major components of the fungal population present in Lascaux Cave, France. The ceiling, walls, sediments and soil were colonized by Fusarium solani in 2001 and later, in 2006, black stains appeared. However, the origin of the successive fungal invasions is unknown as well as the ecology of the cave.
Methods and Results:  The primers nu-SSU-0817F and nu-SSU-1536R were used for the direct amplification of fungal 18S-rDNA sequences from 11 samples. A total of 607 clones were retrieved. Eight out of the ten most abundant phylotypes corresponded to fungi associated with arthropods and represented about 50% of the clones.
Conclusions:  Entomophilous fungi play an important role in the cave and arthropods contribute to the dispersion of spores and fungal development.
Significance and Impact of the Study:  Choosing appropriate targets for control of fungal dispersal is dependent on knowing the causes of fungal colonization. A control of the arthropod populations seems to be a need in order to protect the rock art paintings in Lascaux Cave.     

Radiocarbon--a low-impact tool to study nutrient transport by soil fungi under field conditions

Here, we present a new in-situ method to study the uptake of amino acids by soil fungi. We injected 14C-labeled glycine into a marshland soil and measured the rate and the 14C signature of CO2 respired from sporocarps of Pholiota terrestris over 53.5 h and 2 m. We also determined the incorporation of glycine-C into sporocarp tissue. The 14C signature of the CO2 and tissue was quantified by accelerator mass spectrometry. After the label application, the rate of CO2 flux and its 14C signature from chambers with sporocarps were significantly higher than from chambers without sporocarps, and then declined with time. Postlabel, the 14C signature of the sporocarp tissue increased by 35 per thousand. We show that this approach can be used to study below-ground food webs on an hourly time-scale while minimizing the perturbation of competitive relationships among soil microorganisms and between plants and soil microorganisms. Additionally we show that care must be taken to avoid confounding effects of sporocarp senescence on rates and radiocarbon signatures of respired CO2.     

The number of nuclei in basidiospores of 63 species of ectomycorrhizal Homobasidiomycetes

The production of even a limited number of heterokaryotic spores would be advantageous for establishing new individuals after long distance dispersal. While Suillus and Laccaria species are known to produce binucleate, heterokaryotic spores, this condition is poorly studied for most ectomycorrhizal fungi. To begin addressing this matter the number of nuclei in basidiospores was recorded from 142 sporocarps in 63 species and 20 genera of ectomycorrhizal (EM) fungi. The mean proportion of binucleate basidiospores produced by sporocarps within a species ranged from 0.00 to 1.00, with most genera within a family showing similar patterns. Basidiospores from fungi in Amanita, Cortinariaceae and Laccaria were primarily binucleate but were likely still homokaryotic. Basidiospores from fungi in Boletaceae, Cantharellus, Rhizopogonaceae, Russulaceae, Thelephorales and Tricholoma were primarily uninucleate, but binucleate basidiospores were observed in many genera and in high levels in Boletus. Further research is needed to relate basidiospore nuclear number to reproductive potential in ectomycorrhizal species.     

Environmental filtering affects soil fungal community composition more than dispersal limitation at regional scales

The relative importance of dispersal limitation versus environmental filtering for community assembly has received much attention for macroorganisms. These processes have only recently been examined in microbial communities. Instead, microbial dispersal has mostly been measured as community composition change over space (i.e., distance decay). Here we directly examined fungal composition in airborne wind currents and soil fungal communities across a 40 000 km² regional landscape to determine if dispersal limitation or abiotic factors were structuring soil fungal communities. Over this landscape, neither airborne nor soil fungal communities exhibited compositional differences due to geographic distance. Airborne fungal communities shifted temporally while soil fungal communities were correlated with abiotic parameters. These patterns suggest that environmental filtering may have the largest influence on fungal regional community assembly in soils, especially for aerially dispersed fungal taxa. Furthermore, we found evidence that dispersal of fungal spores differs between fungal taxa and can be both a stochastic and deterministic process. The spatial range of soil fungal taxa was correlated with their average regional abundance across all sites, which may imply stochastic dispersal mechanisms. Nevertheless, spore volume was also negatively correlated with spatial range for some species. Smaller volume spores may be adapted to long-range dispersal, or establishment, suggesting that deterministic fungal traits may also influence fungal distributions. Fungal life-history traits may influence their distributions as well. Hypogeous fungal taxa exhibited high local abundance, but small spatial ranges, while epigeous fungal taxa had lower local abundance, but larger spatial ranges. This study is the first, to our knowledge, to directly sample air dispersal and soil fungal communities simultaneously across a regional landscape. We provide some of the first evidence that soil fungal communities are mostly assembled through environmental filtering and experience little dispersal limitation.     

Secondary chemicals protect mould from fungivory

The vast repertoire of toxic fungal secondary metabolites has long been assumed to have an evolved protective role against fungivory. It still remains elusive, however, whether fungi contain these compounds as an anti-predator adaptation. We demonstrate that loss of secondary metabolites in the soil mould Aspergillus nidulans causes, under the attack of the fungivorous springtail Folsomia candida, a disadvantage to the fungus. Springtails exhibited a distinct preference for feeding on a mutant deleted for LaeA, a global regulator of Aspergillus secondary metabolites. Consumption of the mutant yielded a reproductive advantage to the arthropod but detrimental effects on fungal biomass compared with a wild-type fungus capable of producing the entire arsenal of secondary metabolites. Our results demonstrate that fungal secondary metabolites shape food choice behaviour, can affect population dynamics of fungivores, and suggest that fungivores may provide a selective force favouring secondary metabolites synthesis in fungi.     

Body masses,functional responses and predator–prey stability

The stability of ecological communities depends strongly on quantitative characteristics of population interactions (type‐II vs. type‐III functional responses) and the distribution of body masses across species. Until now, these two aspects have almost exclusively been treated separately leaving a substantial gap in our general understanding of food webs. We analysed a large data set of arthropod feeding rates and found that all functional‐response parameters depend on the body masses of predator and prey. Thus, we propose generalised functional responses which predict gradual shifts from type‐II predation of small predators on equally sized prey to type‐III functional‐responses of large predators on small prey. Models including these generalised functional responses predict population dynamics and persistence only depending on predator and prey body masses, and we show that these predictions are strongly supported by empirical data on forest soil food webs. These results help unravelling systematic relationships between quantitative population interactions and large‐scale community patterns.     

Spatial variability of arbuscular mycorrhizal fungal spores in two natural plant communities

Geostatistical techniques were used to assess the spatial patterns of spores of arbuscular mycorrhizal fungi (AMF) in soils from two contrasting plant communities: a salt marsh containing only arbuscular mycorrhizal and non-mycorrhizal plants in a distinct clumped distribution pattern and a maquis with different types of mycorrhiza where most plants were relatively randomly distributed. Also evaluated was the relationship between the spatial distribution of spores and AM plant distribution and soil properties. A nested sampling scheme was applied in both sites with sample cores taken from nested grids. Spores of AMF and soil characteristics (organic matter and moisture) were quantified in each core, and core sample location was related to plant location. Semivariograms for spore density indicated strong spatial autocorrelation and a patchy distribution within both sites for all AM fungal genera found. However, the patch size differed between the two plant communities and AM fungal genera. In the salt marsh, AM fungal spore distribution was correlated with distance to AM plants and projected stand area of AM plants. In maquis, spatial AM fungal spore distribution was correlated with organic matter. These results suggest that spore distribution of AMF varied between the two plant communities according to plant distribution and soil properties.     

The red imported fire ant,Solenopsis invicta,modifies predation at the soil surface and in cotton foliage

Red imported fire ants, Solenopsis invicta, are generalist predators that can have major impacts on foliar arthropod communities in agricultural systems; however, their effects as predators at the soil surface have not been adequately characterized. We examined the contribution of fire ants to predation at the soil surface and in cotton foliage at two sites and over the course of two field seasons in Georgia, using egg masses of the beet armyworm, Spodoptera exigua. To assess interactions between fire ants and other arthropod species, we also measured the densities of edaphic predators and honeydew‐producing hemipterans at both sites. The sites occurred in different growing regions (Piedmont and Coastal Plain), and allowed us to characterize the importance of fire ants as predators under different climatic and soil conditions. Fire ant suppression decreased egg predation at both field sites, and predation by fire ants at the soil surface was equal to if not greater than that in cotton foliage. However, the impact of fire ants on predation varied between sites, likely due to differences in climate and the composition and activity of the extant arthropod communities. Our study also indicates that fire ant suppression is associated with decreases in the density of honeydew‐producing insects, and increasing abundance of whiteflies on the plants coincided with a decrease in egg predation at the soil surface. This finding suggests the mutualism between ants and whiteflies may lead to a shift in predation intensity from edaphic towards plant‐based food webs.     

The origin of the attine ant-fungus mutualism.

Cultivation of fungus for food originated about 45-65 million years ago in the ancestor of fungus-growing ants (Formicidae, tribe Attini), representing an evolutionary transition from the life of a hunter-gatherer of arthropod prey, nectar, and other plant juices, to the life of a farmer subsisting on cultivated fungi. Seven hypotheses have been suggested for the origin of attine fungiculture, each differing with respect to the substrate used by the ancestral attine ants for fungal cultivation. Phylogenetic information on the cultivated fungi, in conjunction with information on the nesting biology of extant attine ants and their presumed closest relatives, reveal that the attine ancestors probably did not encounter their cultivars-to-be in seed stores (von Ihering 1894), in rotting wood (Forel 1902), as mycorrhizae (Garling 1979), on arthropod corpses (von Ihering 1894) or ant faeces in nest middens (Wheeler 1907). Rather, the attine ant-fungus mutualism probably arose from adventitious interactions with fungi that grew on walls of nests built in leaf litter (Emery 1899), or from a system of fungal myrmecochory in which specialized fungi relied on ants for dispersal (Bailey 1920) and in which the ants fortuitously vectored these fungi from parent to offspring nests prior to a true fungicultural stage. Reliance on fungi as a dominant food source has evolved only twice in ants: first in the attine ants, and second in some ant species in the solenopsidine genus Megalomyrmex that either coexist as trophic parasites in gardens of attine hosts or aggressively usurp gardens from them. All other known ant-fungus associations are either adventitious or have nonnutritional functions (e.g., strengthening of carton-walls in ant nests). There exist no unambiguous reports of facultative mycophagy in ants, but such trophic ant-fungus interactions would most likely occur underground or in leaf litter and thus escape easy observation. Indirect evidence of fungivory can be deduced from contents of the ant alimentary canal and particularly from the contents of the infrabuccal pocket, a pharyngeal device that filters out solids before liquids pass into the intestine. Infrabuccal pocket contents reveal that ants routinely ingest fungal spores and hyphal material. Infrabuccal contents are eventually expelled as a pellet on nest middens or away from the nest by foragers, suggesting that the pellet provides fungi with a means for the dispersal of spores and hyphae. Associations between such "buccophilous" fungi and ants may have originated multiple times and may have become elaborated and externalized in the case of the attine ant-fungus mutualism. Thus, contrary to the traditional model in which attine fungi are viewed as passive symbionts that happened to come under ant control, this alternative model of a myrmecochorous origin of the attine mutualism attributes an important role to evolutionary modifications of the fungi that preceded the ant transition from hunter-gatherer to fungus farmer.