High richness of ectomycorrhizal fungi and low host specificity in a coastal sand dune ecosystem revealed by network analysis

Ectomycorrhizal (EM) fungi are ubiquitous in temperate and boreal forests, comprising over 20,000 species forming root symbiotic associations with Pinaceae and woody angiosperms. As much as 100 different EM fungal species can coexist and interact with the same tree species, forming complex multispecies networks in soils. The degree of host specificity and structural properties of these interaction networks (e.g., nestedness and modularity) may influence plant and fungal community assembly and species coexistence, yet their structure has been little studied in northern coniferous forests, where trees depend on EM fungi for nutrient acquisition. We used high‐throughput sequencing to characterize the composition and diversity of bulk soil and root‐associated fungal communities in four co‐occurring Pinaceae in a relic foredune plain located at Îles de la Madeleine, Québec, Canada. We found high EM fungal richness across the four hosts, with a total of 200 EM operational taxonomic units (OTUs), mainly belonging to the Agaricomycetes. Network analysis revealed an antinested pattern in both bulk soil and roots EM fungal communities. However, there was no detectable modularity (i.e., subgroups of interacting species) in the interaction networks, indicating a low level of specificity in these EM associations. In addition, there were no differences in EM fungal OTU richness or community structure among the four tree species. Limited shared resources and competitive exclusion typically restrict the number of taxa coexisting within the same niche. As such, our finding of high EM fungal richness and low host specificity highlights the need for further studies to determine the mechanisms enabling such a large number of EM fungal species to coexist locally on the same hosts.     

Spatial patterns of plant diversity below-ground as revealed by DNA barcoding

Our understanding of the spatial organization of root diversity in plant communities and of the mechanisms of community assembly has been limited by our ability to identify plants based on root tissue, especially in diverse communities. Here, we test the effectiveness of the plastid gene rbcL, a core plant DNA barcoding marker, for investigating spatial patterns of root diversity, and relate observed patterns to above-ground community structure. We collected 3800 root fragments from four randomly positioned, 1-m-deep soil profiles (two vertical transects per plot), located in an old-field community in southern Ontario, Canada, and extracted and sequenced DNA from 1531 subsampled fragments. We identified species by comparing sequences with a DNA barcode reference library developed previously for the local flora. Nearly 85% of sampled root fragments were successfully sequenced and identified as belonging to 29 plant species or species groups. Root abundance and species richness varied in horizontal space and were negatively correlated with soil depth. The relative abundance of taxa below-ground was correlated with their frequency above-ground (r = 0.73, P = 0.0001), but several species detected in root tissue were not observed in above-ground quadrats. Multivariate analyses indicated that diversity was highly structured below-ground, and associated with depth, root morphology, soil chemistry and soil texture, whereas little structure was evident above-ground. Furthermore, analyses of species co-occurrence indicates strong species segregation overall but random co-occurrence among confamilials. Our results provide insights into the role of environmental filtering and competitive interactions in the organization of plant diversity below-ground, and also demonstrate the utility of barcoding for the identification of plant roots.     

Root-associated ectomycorrhizal fungi shared by various boreal forest seedlings naturally regenerating after a fire in interior alaska and correlation of different fungi with host growth responses

The role of common mycorrhizal networks (CMNs) in postfire boreal forest successional trajectories is unknown. We investigated this issue by sampling a 50-m by 40-m area of naturally regenerating black spruce (Picea mariana), trembling aspen (Populus tremuloides), and paper birch (Betula papyrifera) seedlings at various distances from alder (Alnus viridis subsp. crispa), a nitrogen-fixing shrub, 5 years after wildfire in an Alaskan interior boreal forest. Shoot biomasses and stem diameters of 4-year-old seedlings were recorded, and the fungal community associated with ectomycorrhizal (ECM) root tips from each seedling was profiled using molecular techniques. We found distinct assemblages of fungi associated with alder compared with those associated with the other tree species, making the formation of CMNs between them unlikely. However, among the spruce, aspen, and birch seedlings, there were many shared fungi (including members of the Pezoloma ericae [Hymenoscyphus ericae] species aggregate, Thelephora terrestris, and Russula spp.), raising the possibility that these regenerating seedlings may form interspecies CMNs. Distance between samples did not influence how similar ECM root tip-associated fungal communities were, and of the fungal groups identified, only one of them was more likely to be shared between seedlings that were closer together, suggesting that the majority of fungi surveyed did not have a clumped distribution across the small scale of this study. The presence of some fungal ribotypes was associated with larger or smaller seedlings, suggesting that these fungi may play a role in the promotion or inhibition of seedling growth. The fungal ribotypes associated with larger seedlings were different between spruce, aspen, and birch, suggesting differential impacts of some host-fungus combinations. One may speculate that wildfire-induced shifts in a given soil fungal community could result in variation in the growth response of different plant species after fire and a shift in regenerating vegetation.     

Characteristics and abundance of vertebrate-dispersed fruits in temperate wet sclerophyll forest in southeastern Australia

The diversity, abundance and display characteristics of plants producing fleshy fruits were recorded in wet sclerophyll forest near Melbourne, Victoria. Fruit characteristics and nutrient content were recorded for 14 species of fruits occurring in the forest and these characteristics compared with similar studies in north temperate and tropical regions. Plants producing fleshy fruits were confined to understorey layers and comprised 13.5% of the native vascular flora. Most species displayed fruits conspicuously by ripening brightly coloured fruits synchronously in large displays. Annual fruit production at three sites ranged from 6.98 to 37.01 kg/ha. Fruit mass, seed number and size were variable but the proportion of pulp per fruit remained remarkably consistent between species. Most fruit pulp contained high quantities of soluble carbohydrates and little lipid or protein. Fruits from wet sclerophyll forest did not differ from fruits from other geographic areas in eight characteristics measured. Although taxonomically distinct, the species in wet sclerophyll forest were not significantly different from other temperate fruits, suggesting that fruit characteristics are under the influence of factors other than phylogeny.     

DNA barcoding as a complementary tool for conservation and valorisation of forest resources

Since the pre-historic era, humans have been using forests as a food, drugs and handcraft reservoir. Today, the use of botanical raw material to produce pharmaceuticals, herbal remedies, teas, spirits, cosmetics, sweets, dietary supplements, special industrial compounds and crude materials constitute an important global resource in terms of healthcare and economy. In recent years, DNA barcoding has been suggested as a useful molecular technique to complement traditional taxonomic expertise for fast species identification and biodiversity inventories. In this study, in situ application of DNA barcodes was tested on a selected group of forest tree species with the aim of contributing to the identification, conservation and trade control of these valuable plant resources.The “core barcode” for land plants (rbcL, matK, and trnH-psbA) was tested on 68 tree specimens (24 taxa). Universality of the method, ease of data retrieval and correct species assignment using sequence character states, presence of DNA barcoding gaps and GenBank discrimination assessment were evaluated. The markers showed different prospects of reliable applicability. RbcL and trnH-psbA displayed 100% amplification and sequencing success, while matK did not amplify in some plant groups. The majority of species had a single haplotype. The trnH-psbA region showed the highest genetic variability, but in most cases the high intraspecific sequence divergence revealed the absence of a clear DNA barcoding gap. We also faced an important limitation because the taxonomic coverage of the public reference database is incomplete. Overall, species identification success was 66.7%.This work illustrates current limitations in the applicability of DNA barcoding to taxonomic forest surveys. These difficulties urge for an improvement of technical protocols and an increase of the number of sequences and taxa in public databases.     

Recognition and characterization of four Thai xylariaceous fungi inhabiting various tropical foliages as endophytes by DNA sequences and host plant preference

A total of 202 strains of xylariaceous fungi (183 endophytic strains isolated from 25 plant species of 24 genera in 21 families and 19 saprobic strains), segregated into four clades, were examined by nuclear rDNA internal transcribed spacer (ITS) sequence and beta-tubulin coding region analyses to clarify their taxonomic status and species boundaries. Three of the four species clades were assigned to Xylaria cubensis (100 strains), Xylaria grammica (33 strains), and Nemania diffusa (48 strains). Another fungus was tentatively assigned to Nemania cf. bipapillata (21 strains). Comparison of the host plants revealed that X. cubensis inhabited healthy leaves of at least 24 plant species (23 genera of 21 families) as endophytes; N. diffusa was found on 19 plant species (18 genera of 15 families), Nemania cf. bipapillata on 11 species (10 genera of 9 families), and X. grammica on 8 species (8 genera of 7 families). The present results suggest that the major xylariaceous endophytes in tropical plants are likely to be non-host specific, or have a wide range of host plant preferences.     

Ericoid mycorrhizal fungi are common root inhabitants of non-Ericaceae plants in a south-eastern Australian sclerophyll forest

Fungi were isolated from the roots of 17 plant species from the families Apiaceae, Cunoniaceae, Cyperaceae, Droseraceae, Fabaceae-Mimosoideae, Lomandraceae, Myrtaceae, Pittosporaceae, Proteaceae and Stylidiaceae at a sclerophyll forest site in New South Wales, Australia. Internal transcribed spacer (ITS) restriction fragment length polymorphism (RFLP) and sequence comparisons indicated that the isolated fungi had affinities to a range of ascomycetes, basidiomycetes and zygomycetes. Four RFLP types had closest affinities to previously identified Helotiales ericoid mycorrhizal (ERM) or Oidiodendron spp. Isolates representing six RFLP types, which were variously isolated from all 17 plant species, formed ERM coils in hair root epidermal cells of Woollsia pungens (Ericaceae) under gnotobiotic conditions. Three of these isolates formed intercellular hyphae, intracellular hyphae and/or microsclerotia, which are typical of dark septate endophyte infection, in roots of Stylidium productum (Stylidiaceae), indicating an ability to form different types of association with roots of different hosts. Overall the data indicate that a broad range of plant taxa may act as repositories for ERM fungi in sclerophyll forest soil.     

Diversity and community composition of ectomycorrhizal fungi in a dry deciduous dipterocarp forest in Thailand

Large forest areas of South-East Asia, are dominated by the Dipterocarpaceae tree family, which contains many important timber species. Unlike many other tropical trees, Dipterocarpaceae rely on ectomycorrhizal (ECM) root symbiosis for their mineral nutrition. This study aims to document the richness and community composition of ECM fungi in a dry deciduous forest in Thailand. Combining morphological and molecular identification methods revealed 69 species of ECM fungi that belong to 17 phylogenetic lineages. The /russula-lactarius, /tomentella-thelephora, /sordariales, /sebacina and /cantharellus lineages were the most species-rich. The fungal richness is comparable to other tropical rain forest sites, but the phylogenetic community structure has elements of both tropical and temperate ecosystems. Unlike tropical rain forests, the Cenococcum geophilum complex was one of the most frequent fungal taxa that had a relatively high ITS genetic diversity over the small sampling area. This study provides the first snapshot insight into the fungal community of dry dipterocarp forests. However, it is necessary to broaden the spatial and temporal scales of sampling to improve our understanding of the below-ground relations of dry and humid tropical forests.     

Solubilisation and colonisation of wood ash by ectomycorrhizal fungi isolated from a wood ash fertilised spruce forest

In Sweden application of granulated wood ash has been suggested as a method to supplement nutrient loss resulting from harvesting of forest residues for bioenergy production. Mycelia of two ectomycorrhizal fungi Piloderma sp. 1 and Ha-96-3, were commonly found to colonise ash granules in a wood ash fertilised spruce forest. Thirty-eight fungal isolates were selected from 10 taxa to investigate the possible role of different ectomycorrhizal fungi in nutrient mobilisation from ash. The taxa were Cenococcum geophilum Fr., Piloderma croceum Erikss. and Hjortst., Piloderma sp. 1, Thelephora terrestris (Ehrenb.) Fr., Tylospora fibrillosa Donk, and five unidentified species, all originating from a wood ash fertilised spruce forest. The isolates were tested for their ability to solubilise tricalcium phosphate (TCP) or hardened wood ash (HWA) in vitro. Ha-96-3, P. croceum and Piloderma sp. 1 were the only taxa which solubilised TCP. Abundant calcium oxalate crystals were formed in TCP and HWA plates with Piloderma sp. 1. Ha-96-3 and two isolates of P. croceum produced intermediate amounts of crystals. Ha-96-1 and T. fibrillosa produced low amounts of crystal but no crystal formation was observed by any of the other isolates. Piloderma sp. 1 from HWA plates had significantly higher concentrations of P, compared to P. croceum or Ha-96-3. Piloderma sp. 1 and P. croceum were further tested for their ability to colonise wood ash in microcosms containing intact mycorrhizal associations. After 7 months Piloderma sp. 1 colonised ash amended patches with a dense, mat like mycelium, whereas P. croceum mycelia avoided the ash patches. Possible differences between these fungi in patterns of carbon allocation were investigated by labelling seedlings with 14CO(2). Piloderma sp. 1 mycelia allocated significantly more 14C to ash patches than P. croceum. P. croceum allocated relatively more 14C to control patches than to the ash patches. The possible role of ectomycorrhizal fungi in mobilisation of nutrients from wood ash is discussed.     

New insights on diet variability revealed by DNA barcoding and high-throughput pyrosequencing: chamois diet in autumn as a case study

Characterizing the diet of large herbivores and the determinants of its variation remains a difficult task in wild species. DNA-based techniques have the potential to complement traditional time-consuming methods based on the microhistology of plant cuticle fragments in fecal or rumen samples. Recently, it has been shown that a short chloroplast DNA fragment, the P6 loop of the trnL (UAA) intron, can act as a minimalist barcode. Here, we used the trnL approach with high-throughput pyrosequencing to study diet from feces in a wild herbivore, the alpine chamois (Rupicapra rupicapra) and showed that the fine resolution in plant determination obtained with this method allows exploring subtle temporal shifts and inter-individual variability in diet composition. First, we built a DNA barcoding database of 475 plants species. Seventy-two percent of plant species can be unambiguously identified to species level, 79% to genus level and 100% to family level using the P6 loop. Second, we analysed 74 feces collected from October to November. Based on 47,896 P6 loop sequences, we identified a total of 110 taxa, 96 in October and 76 in November, with a clear diet shift between October and November. We recognized four and two clusters of feces composition in October and November, respectively, revealing different diet categories among individuals within each month. DNA-based diet analysis is faster and more taxonomically precise than studies based on microhistology, and opens new possibilities for analysing plant-herbivore interactions in the wild.     

DNA barcoding of lichenized fungi demonstrates high identification success in a floristic context

? Efforts are currently underway to establish a standard DNA barcode region for fungi; we tested the utility of the internal transcribed spacer (ITS) of nuclear ribosomal DNA for DNA barcoding in lichen-forming fungi by sampling diverse species across eight orders. ? Amplification of the ITS region (ITS1-5.8S-ITS2) was conducted for 351 samples, encompassing 107, 55 and 28 species, genera and families, respectively, of lichenized fungi. We assessed the ability of the entire ITS vs the ITS2 alone to discriminate between species in a taxonomic dataset (members of the genus Usnea) and a floristic dataset. ? In the floristic dataset, 96.3% of sequenced samples could be assigned to the correct species using ITS or ITS2; a barcode gap for ITS is present in 92.1% of species. Although fewer species have a barcode gap in the taxonomic dataset (73.3% with ITS and 68.8% with ITS2), up to 94.1% of samples were assigned to the correct species using BLAST. ? While discrimination between the most closely related species will remain challenging, our results demonstrate the potential to identify a high percentage of specimens to the correct species, and the remainder to the correct genus, when using DNA barcoding in a floristic context.     

Diversity and species distribution of ectomycorrhizal fungi along productivity gradients of a southern boreal forest

Coniferous forests with diverse ectomycorrhizal fungus (EMF) communities are associated with nutrient-poor, acidic soils but there is some debate whether EMF can be equally adapted to more productive, nitrogen-rich sites. We compared EMF species distribution and diversity along a replicated productivity gradient in a southern boreal forest of British Columbia (Canada). Roots from subalpine fir (Abies lasiocarpa) saplings of the understory were sampled and EMF species were identified by morphotypes supplemented with ITS rDNA analysis. There were significant changes in the distribution and abundance of 74 EMF species along the productivity gradient, with as little as 24% community similarity among contrasting sites. Species richness per plot increased asymptotically with foliar nitrogen concentrations of subalpine fir, demonstrating that many EMF species were well suited to soils with high rates of nitrogen mineralization. EMF species abundance in relation to site productivity included parabolic, negative linear, and positive exponential curves. Both multi-site and more narrowly distributed EMF were documented, and a diverse mix of mantle exploration types was present across the entire productivity gradient. The results demonstrate strong associations of EMF fungal species with edaphic characteristics, especially nitrogen availability, and a specialization in EMF communities that may contribute to the successful exploitation of such contrasting extremes in soil fertility by a single tree host.     

Diversity and specificity of ectomycorrhizal fungi retrieved from an old-growth Mediterranean forest dominated by Quercus ilex

We analysed the ectomycorrhizal (ECM) fungal diversity in a Mediterranean old-growth Quercus ilex forest stand from Corsica (France), where Arbutus unedo was the only other ECM host. On a 6400 m2 stand, we investigated whether oak age and host species shaped below-ground ECM diversity. Ectomycorrhizas were collected under Q. ilex individuals of various ages (1 yr seedlings; 3-10 yr saplings; old trees) and A. unedo. They were typed by ITS-RFLP analysis and identified by match to RFLP patterns of fruitbodies, or by sequencing. A diversity of 140 taxa was found among 558 ectomycorrhizas, with many rare taxa. Cenococcum geophilum dominated (35% of ECMs), as well as Russulaceae, Cortinariaceae and Thelephoraceae. Fungal species richness was comparable above and below ground, but the two levels exhibited < 20% overlap in fungal species composition. Quercus ilex age did not strongly shape ECM diversity. The two ECM hosts, A. unedo and Q. ilex, tended to share few ECM species (< 15% of the ECM diversity). Implications for oak forest dynamics are discussed.     

Invasion potential and host shifts of Australian and African ectomycorrhizal fungi in mixed eucalypt plantations

? Transportation of forestry materials results in unintended co-introduction of nonnative species that may cause enormous ecological or economic damage. While the invasion ecology of plants and animals is relatively well-known, that of microorganisms, except aboveground pathogens, remains poorly understood. ? This work addresses host shifts and invasion potential of root symbiotic ectomycorrhizal fungi that were co-introduced with Australian eucalypts and planted in clear-cut miombo woodlands in Zambia, south-central Africa. ? By use of rDNA and plastid intron sequence analysis for identification and phylogenetic techniques for inferring fungal origin, we demonstrated that host shifts were uncommon in the Australian fungi, but frequent in the African fungi, especially in mixed plantations where roots of different trees intermingle. ? There was evidence for naturalization, but not for invasion by Australian ectomycorrhizal fungi. Nevertheless, the fungi introduced may pose an invasion risk along with further adaptation to local soil environment and host trees. Inoculation of eucalypts with native edible fungi may ameliorate the potential invasion risks of introduced fungi and provide an alternative source of nutrition.     

Community structure of ectomycorrhizal fungi in a Pinus muricata forest: minimal overlap between the mature forest and resistant propagule communities

We have investigated colonization strategies by comparing the abundance and frequency of ectomycorrhizal fungal species on roots in a mature Pinus muricata forest with those present as resistant propagules colonizing potted seedlings grown in the same soil samples. Thirty-seven fungal species were distinguished by internal transcribed spacer (ITS) restriction fragment length polymorphisms (RFLPs); most were identified to species level by sporocarp RFLP matches or to genus/family level by using sequence databases for the mitochondrial and nuclear large-subunit rRNA genes. The below-ground fungal community found in the mature forest contrasted markedly with the resistant propagule community, as only four species were found in both communities. The dominant species in the mature forest were members of the Russulaceae, Thelephorales and Amanitaceae. In contrast, the resistant propagule community was dominated by Rhizopogon species and by species of the Ascomycota. Only one species, Tomentella sublilacina (Thelephorales), was common in both communities. The spatial distribution of mycorrhizae on mature roots and propagules in the soil differed among the dominant species. For example, T. sublilacina mycorrhizae exhibited a unique bias toward the organic horizons, Russula brevipes mycorrhizae were denser and more clumped than those of other species and Cenococcum propagules were localized, whereas R. subcaerulescens propagules were evenly distributed. We suggest that species differences in resource preferences and colonization strategies, such as those documented here, contribute to the maintenance of species richness in the ectomycorrhizal community.     

Cryptic diversity in Indo-Australian rainbowfishes revealed by DNA barcoding: implications for conservation in a biodiversity hotspot candidate

The rainbowfishes of the family Melanotaeniidae represent one of the largest radiations of freshwater fishes from the Indo-Australian archipelago. A total of 75 nominal species have been described, among which several have become very popular among tropical fish hobbyists because of their tendency to form large schools of colourful individuals. Facing habitat loss and competition or predation by introduced species, this group has become a priority in the conservation of ornamental fishes in Indonesia. In this context, several expeditions have been conducted between 2007 and 2010 in Indonesian Papua with the aim to initiate a large-scale survey of the genetic resources in this group. We assessed the diversity of the Papua rainbowfishes with DNA barcoding. We sequenced the mitochondrial COI gene for 350 specimens belonging to 53 nominal species throughout the Indo-Australian archipelago. Unexpected levels of cryptic diversity and endemism were detected since additional cryptic lineages were detected in several watersheds from the Vogelkop and the Lengguru massif. DNA barcoding supports the presence of nearly 30 evolutionary lineages among the 15 nominal species sampled in the Vogelkop and all these lineages are endemic to a single lake or watershed. This result highlights that the diversity of the family has been largely underestimated and urges for the identification of conservation priorities in Papua.