Combining high-throughput sequencing with fruit body surveys reveals contrasting life-history strategies in fungi

Before the recent revolution in molecular biology, field studies on fungal communities were mostly confined to fruit bodies, whereas mycelial interactions were studied in the laboratory. Here we combine high-throughput sequencing with a fruit body inventory to study simultaneously mycelial and fruit body occurrences in a community of fungi inhabiting dead wood of Norway spruce. We studied mycelial occurrence by extracting DNA from wood samples followed by 454-sequencing of the ITS1 and ITS2 regions and an automated procedure for species identification. In total, we detected 198 species as mycelia and 137 species as fruit bodies. The correlation between mycelial and fruit body occurrences was high for the majority of the species, suggesting that high-throughput sequencing can successfully characterize the dominating fungal communities, despite possible biases related to sampling, PCR, sequencing and molecular identification. We used the fruit body and molecular data to test hypothesized links between life history and population dynamic parameters. We show that the species that have on average a high mycelial abundance also have a high fruiting rate and produce large fruit bodies, leading to a positive feedback loop in their population dynamics. Earlier studies have shown that species with specialized resource requirements are rarely seen fruiting, for which reason they are often classified as red-listed. We show with the help of high-throughput sequencing that some of these species are more abundant as mycelium in wood than what could be expected from their occurrence as fruit bodies.     

Analysis of macrofungal communities reveals a complex reciprocal influence between Mediterranean montane calcareous grassland and surrounding forest habitats

Fungi are essential components of all terrestrial ecosystems. Despite the crucial ecological role of soil fungi in grasslands, knowledge about fungal community diversity and structure in Mediterranean meadow habitats is still fragmentary. We analyzed macrofungal communities in three geographically distinct Mediterranean montane calcareous grasslands and surrounding forests, by means of fruit body surveys. We investigated a number of biotic and abiotic factors influencing the studied fungal communities, including plant species composition. Out of 6365 fruit bodies, a total of 268 species belonging to 84 genera were found. In general, there was a significant correlation between plant species richness and fungal richness. Variation in vegetation and plant community structure accounted for approximately 20% of variance in fungal community structure. Tree and shrub vegetation played a dominant role in shaping the analyzed fungal communities, both in meadows and surrounding forests, with particular influence on ectomycorrhizal, litter, and lignicolous saprotrophic fungi. Fungal biodiversity in the studied meadows was increased by the presence of tree and shrub species from the adjacent forests, but was reduced by the increasing vegetation cover.     

Taxonomic gap in wood-inhabiting fungi: identifying understudied groups by a systematic survey

Despite advances in phylogenetic research and the number of ecological studies focusing on wood-inhabiting fungi, these species still represent a taxonomically poorly known group of organisms. In this study, our overall aim was to detect and characterize the understudied wood-inhabiting fungal groups in the beech forests of Navarre (northern Spain). We present a list of 326 wood-inhabiting fungal species, out of which 36 % are first regional records. Comparing the already recorded fungal species in this territory and the list of firstly recorded species, we found that field-mycologists tend to focus on certain fungal groups, and in general rare species are less frequently encountered. Particularly, species with corticioid fruit body type have been especially overlooked in this territory. We attribute the high proportion of new regional records to the use of a systematic sampling design.     

Diversity of basidiomycetes in michigan agricultural soils

We analyzed the communities of soil basidiomycetes in agroecosystems that differ in tillage history at the Kellogg Biological Station Long-Term Ecological Research site near Battle Creek, Michigan. The approach combined soil DNA extraction through a bead-beating method modified to increase recovery of fungal DNA, PCR amplification with basidiomycete-specific primers, cloning and restriction fragment length polymorphism screening of mixed PCR products, and sequencing of unique clones. Much greater diversity was detected than was anticipated in this habitat on the basis of culture-based methods or surveys of fruiting bodies. With "species" defined as organisms yielding PCR products with > or =99% identity in the 5' 650 bases of the nuclear large-subunit ribosomal DNA, 241 "species" were detected among 409 unique basidiomycete sequences recovered. Almost all major clades of basidiomycetes from basidiomycetous yeasts and other heterobasidiomycetes through polypores and euagarics (gilled mushrooms and relatives) were represented, with a majority from the latter clade. Only 24 of 241 "species" had 99% or greater sequence similarity to named reference sequences in GenBank, and several clades with multiple "species" could not be identified at the genus level by phylogenetic comparisons with named sequences. The total estimated "species" richness for this 11.2-ha site was 367 "species" of basidiomycetes. Since >99% of the study area has not been sampled, the accuracy of our diversity estimate is uncertain. Replication in time and space is required to detect additional diversity and the underlying community structure.     

Species associations during the succession of wood-inhabiting fungal communities

We studied fungal succession in decaying wood by compiling time-series data of fruit body observations. We tested the hypothesis that the presence of a primary species affects the probability of a succeeding species occurring later on the same log. Significant associations were detected for 15 species pairs; these were consistent with earlier findings on co-occurrence patterns in single time surveys. We used enrichment analysis to test if species with particular life-history attributes were more often associated with the occurrence of a succeeding species, or vice versa. White rot fungi and fungi abundant as mycelia were more often associated with the occurrence of succeeding species, compared to brown rot fungi and species with low mycelial abundance. Our results indicate that certain primary species cause priority effects and non-random co-occurrence patterns in the field. These successional patterns are likely to be connected both with substrate modification and species interactions.     

Polypore diversity in North America with an annotated checklist

Profound changes to the taxonomy and classification of polypores have occurred since the advent of molecular phylogenetics in the 1990s. The last major monograph of North American polypores was published by Gilbertson and Ryvarden in 1986–1987. In the intervening 30 years, new species, new combinations, and new records of polypores were reported from North America. As a result, an updated checklist of North American polypores is needed to reflect the polypore diversity in there. We recognize 492 species of polypores from 146 genera in North America. Of these, 232 species are unchanged from Gilbertson and Ryvarden’s monograph, and 175 species required name or authority changes. In addition, 40 new species and 45 new records published since that monograph are included in the checklist. Among the 492 species of polypores, 486 species from 143 genera belong to 11 orders, while six other species from three genera have uncertain taxonomic position at the order level. Three orders, viz. Polyporales, Hymenochaetales and Russulales, accommodate most of polypore species (93.7 %) and genera (88.8 %). We hope that this updated checklist will inspire future studies in the polypore mycota of North America and contribute to the diversity and systematics of polypores worldwide.     

Correspondence between genet diversity and spatial distribution of above- and below-ground populations of the ectomycorrhizal fungus Hebeloma cylindrosporum

Population studies of ectomycorrhizal fungal species have largely relied upon fruit body (the reproductive organ) sampling. Analysis of the fruit bodies alone supposes that they reflect the present and spatial organization of all below-ground genets (mycorrhizas and extramatrical mycelia). The relation between fruit bodies and ectomycorrhizas was investigated for the basidiomycete agaric Hebeloma cylindrosporum in four Pinus pinaster stands in south-west France. Genet identification was based on the comparison of polymorphisms within a hypervariable segment of the ribosomal intergenic spacer amplified by polymerase chain reaction (PCR) using a H. cylindrosporum species-specific primer. Mycorrhizas were sorted from soil samples collected underneath patches of fruit bodies or patches where fruit bodies had or had not been observed during the years prior to mycorrhiza collection. On average 65% of the 1026 mycorrhizas collected underneath fruit bodies were formed by H. cylindrosporum, whereas only 2% of the 954 collected in places from where fruit bodies were absent were formed by this species. All genotypes identified above ground were also identified below ground. In patches where one genotype formed all or more than 90% of the fruit bodies, the same genotype formed all or a large majority of the mycorrhizas. In patches occupied by several different fruiting genotypes, additional nonfruiting ones could be present on the root systems. In all cases, the mycorrhizas of one genotype were found no more than 10-20 cm away from its corresponding fruit bodies, and fruit body disappearance at a given place was associated with the disappearance of the corresponding mycorrhizas within 1 year. Although there was not a strict coincidence between the total numbers of genets present below ground and of those forming fruit bodies, fruit body analysis for H. cylindrosporum appears to reflect both the genetic diversity and the spatial structure of its below-ground populations. The results obtained also illustrate the rapid turnover of ectomycorrhizal fungal species on the root systems in the absence of any obvious major disturbance of the ecosystem.     

Fruiting body and soil rDNA sampling detects complementary assemblage of Agaricomycotina (Basidiomycota, Fungi) in a hemlock-dominated forest plot in southern Ontario

This is the first study to assess the diversity and community structure of the Agaricomycotina in an ectotrophic forest using above-ground fruiting body surveys as well as soil rDNA sampling. We recovered 132 molecular operational taxonomic units, or 'species', from fruiting bodies and 66 from soil, with little overlap. Fruiting body sampling primarily recovered fungi from the Agaricales, Russulales, Boletales and Cantharellales. Many of these species are ectomycorrhizal and form large fruiting bodies. Soil rDNA sampling recovered fungi from these groups in addition to taxa overlooked during the fruiting body survey from the Atheliales, Trechisporales and Sebacinales. Species from these groups form inconspicuous, resupinate and corticioid fruiting bodies. Soil sampling also detected fungi from the Hysterangiales that form fruiting bodies underground. Generally, fruiting body and soil rDNA samples recover a largely different assemblage of fungi at the species level; however, both methods identify the same dominant fungi at the genus-order level and ectomycorrhizal fungi as the prevailing type. Richness, abundance, and phylogenetic diversity (PD) identify the Agaricales as the dominant fungal group above- and below-ground; however, we find that molecularly highly divergent lineages may account for a greater proportion of total diversity using the PD measure compared with richness and abundance. Unless an exhaustive inventory is required, the rapidity and versatility of DNA-based sampling may be sufficient for a first assessment of the dominant taxonomic and ecological groups of fungi in forest soil.     

Fungal Community Analysis by Large-Scale Sequencing of Environmental Samples

Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized.     

Growth sites of polypores from quantitative expert evaluation: Late-stage decayers and saprotrophs fruit closer to ground

Life history traits are key to why species occur when and where they do and how their populations will respond to environmental changes. However, dispersal-related traits of fungi are generally poorly known. We studied how spore release height from the ground, an important determinant of airborne dispersal, is connected to other traits in polypores. We collected expert evaluations of fruit body growth sites for 140 species and found that experts generally provided consistent estimates of height above the ground. Height was correlated with other traits: species fruiting on living trees, earlier decay stages and deciduous hosts tend to fruit higher above the ground. While our data do not allow mechanistic explanations, our study demonstrates the potential of expert knowledge and identifies fruit body height above the ground as one consistent trait relevant to species’ life history strategies. We recommend a more comprehensive expert survey as one cost-efficient way towards a more trait-based fungal ecology.     

Fungal community analysis by large-scale sequencing of environmental samples

Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized. All sequences were deposited in GenBank, with accession numbers AY 969316 to AY 970290 for the ITS sequences and AY 969135 to AY 969315 for the SSU sequences.     

High-throughput sequencing of amplicons for monitoring yeast biodiversity in must and during alcoholic fermentation

We compared pyrosequencing technology with the PCR-ITS-RFLP analysis of yeast isolates and denaturing gradient gel electrophoresis (DGGE). These methods gave divergent findings for the yeast population. DGGE was unsuitable for the quantification of biodiversity and its use for species detection was limited by the initial abundance of each species. The isolates identified by PCR-ITS-RFLP were not fully representative of the true population. For population dynamics, high-throughput sequencing technology yielded results differing in some respects from those obtained with other approaches. This study demonstrates that 454 pyrosequencing of amplicons is more relevant than other methods for studying the yeast community on grapes and during alcoholic fermentation. Indeed, this high-throughput sequencing method detected larger numbers of species on grapes and identified species present during alcoholic fermentation that were undetectable with the other techniques.     

Is fungal species richness and composition related to the occurrence of the old‐growth associated wood‐decaying Amylocystis lapponica?

Amylocystis lapponica (Romell) Singer is a widely distributed wood‐decaying polypore fungus found throughout the Northern Hemisphere. Despite its huge distribution range it occurs rather patchily and seems narrowly associated with old‐growth forest stands. Notably, it has been used as an ‘indicator species’, believed to reflect the long‐term presence of dead wood, naturalness of forest stands, and indirectly, species richness and possibly composition. In this study we focused on the last issue – whether or not there is a link between the occurrence of A. lapponica and the species richness and composition of other wood‐decaying fungi. Selecting log characteristics and microclimate as similar as possible, we compared 12 logs with and 12 logs without visible fruit bodies of A. lapponica to examine: 1) if visible fruit bodies corresponded with molecular identification of the mycelia, 2) if fungal species richness and composition of the substrate were related to A. lapponica occurrence, and 3) if A. lapponica was restricted to certain parts of the log. Fungal species were recorded by inspecting visible fruit bodies and by culture isolation and ITS sequencing from wood disc samples. Laboratory and field identification of A. lapponica had 71% correspondence, and mycelia were identified in two logs without visible fruit bodies. Twice as many fungal species were detected using ITS sequencing compared to fruit body identification. Total species richness was similar between the two log categories, but number of species per log was slightly higher in A. lapponica logs. Antrodia serialis (Fr.) Donk, and possibly also Fomitopsis pinicola (Sw.:Fr.) P. Karst. and Phellinus nigrolimitatus (Romell) Bourdot & Galzin, occurred more frequently in A. lapponica logs. Mycelia of A. lapponica were restricted to less decayed parts of the wood in the centre of the middle part of the logs.     

Perennial polypores as indicators of annual and red-listed polypores

Many polypores are specialized in their requirements for substrate and environment, and they have been suggested to indicate the continuity of coarse woody debris or naturalness of a forest stand. However, the use of polypores as indicators of conservation value is restricted by the temporally limited appearance of annual fruit bodies. We studied whether the species richness of perennial polypores (perennials) can be used to predict the species richness of annual or annual red-listed polypores (annuals). Our data included 1471 separate datasets (sample plots or larger inventoried areas) in different parts of Finland and Russian Karelia, ranging from the southern to northern boreal zone. At the large scale (the whole area) the number of perennials explained about 70% of the variation in the number of annuals, and about 67% in the number of red-listed annuals. A minimum set of 40–60 perennial occurrences gave a reliable estimate on the species richness of annuals, and 60–80 occurrences on the species richness of red-listed annuals. The richness of perennials predicted the richness of annuals and, in particular, richness of red-listed annuals, better than the size of inventoried area. According to our results, perennial polypores can be used as a surrogate for overall polypore species richness in natural and seminatural boreal forests, but the predictive power is weaker in managed forests. In addition, the relationship between the perennial and annual species seems to differ in different vegetation zones, management types and forest types. Due to this variation direct application of the indicator values derived from different vegetation zones and management or forest types are not recommended. Since perennials are easier to identify than annuals, detectable throughout the year, and have much smaller year-to-year variation, their use as an indicator group seems to offer advantages regarding the timing and cost-efficiency of inventories.     

Fallen retention aspen trees on clear-cuts can be important habitats for red-listed polypores: a case study in Finland

Green-tree retention is a relatively new forestry application, which aims at decreasing the negative effects of clear-cut logging on forest biodiversity. In this study, the value of retained aspens in maintaining diverse assemblages of wood-decaying fungi (polypores; Basidiomycota) on clear-cuts was investigated, after the retention trees had died, fallen and started to decay. A total of 110 fallen aspen trunks were investigated on clear-cuts and within old-growth forests in eastern Finland, southern boreal zone; and 499 records of polypores belonging to 46 species were made. The intermediately decayed trunks on a clear-cut area hosted more species and more red-listed species than did trunks within forests. Most of the polypore species with more than two records were found in both habitats. These results suggest that many aspen-associated polypores are able to live and reproduce in sun-exposed habitats, if the quality and quantity of dead wood fulfill the species-specific requirements. This unexpected result, however, may be partly due to the exceptionally great abundance of aspen in the study area. Furthermore, in the long term, the local benefits of fallen retention trees can be limited, unless the local continuity of large aspens, both living and dead, is ensured.     

Effort versus Reward: Preparing Samples for Fungal Community Characterization in High-Throughput Sequencing Surveys of Soils

Next generation fungal amplicon sequencing is being used with increasing frequency to study fungal diversity in various ecosystems; however, the influence of sample preparation on the characterization of fungal community is poorly understood. We investigated the effects of four procedural modifications to library preparation for high-throughput sequencing (HTS). The following treatments were considered: 1) the amount of soil used in DNA extraction, 2) the inclusion of additional steps (freeze/thaw cycles, sonication, or hot water bath incubation) in the extraction procedure, 3) the amount of DNA template used in PCR, and 4) the effect of sample pooling, either physically or computationally. Soils from two different ecosystems in Minnesota, USA, one prairie and one forest site, were used to assess the generality of our results. The first three treatments did not significantly influence observed fungal OTU richness or community structure at either site. Physical pooling captured more OTU richness compared to individual samples, but total OTU richness at each site was highest when individual samples were computationally combined. We conclude that standard extraction kit protocols are well optimized for fungal HTS surveys, but because sample pooling can significantly influence OTU richness estimates, it is important to carefully consider the study aims when planning sampling procedures.     

Performance of ground-based and aerial survey methods for monitoring wildlife assemblages in a conservation area of northern Tanzania

Validating and improving field-sampling techniques for estimating wildlife community composition and population size is essential for wildlife management and conservation. We conducted ground distance sampling surveys along line transects and block counts from a small aircraft in Manyara Ranch in Northern Tanzania and contrasted estimates of species richness and species-specific densities from both sampling techniques. We used regression analyses (logistic regression and generalized linear mixed models) and model selection to investigate whether a species’ body size, group size, body color, as well as vegetation cover explained the variation in species presence/absence and relative density differences in aerial vs. ground-based sampling. Ground surveys detected significantly more species than aerial surveys. However, aerial surveys detected three species that were missed by ground surveys (African lions, African buffalo, and spotted hyena). Model selection suggested that species with smaller body mass and small group sizes were more likely to be missed in aerial surveys. Densities estimated from the aerial surveys were generally but non-significantly lower than the densities estimated from the ground surveys, with the exception of density estimates for African elephants which were slightly higher from aerial surveys. Density differences between the two methods were greater for species with small group size, light body color, and in areas with denser vegetation cover; these variables explained 75% of the variation in density differences between the two survey methods. Albeit being similar in operational costs in our relatively small study area, ground surveys yielded (1) more complete information with respect to wildlife community composition and (2) density estimates were mostly higher and (3) more precise and (4) appear more feasible to be implemented in community-based conservation schemes.     

Carrion fly‐derived DNA metabarcoding is an effective tool for mammal surveys: Evidence from a known tropical mammal community

Metabarcoding of vertebrate DNA derived from carrion flies has been proposed as a promising tool for biodiversity monitoring. To evaluate its efficacy, we conducted metabarcoding surveys of carrion flies on Barro Colorado Island (BCI), Panama, which has a well‐known mammal community, and compared our results against diurnal transect counts and camera trapping. We collected 1,084 flies in 29 sampling days, conducted metabarcoding with mammal‐specific (16S) and vertebrate‐specific (12S) primers, and sequenced amplicons on Illumina MiSeq. For taxonomic assignment, we compared blast with the new program protax , and we found that protax improved species identifications. We detected 20 mammal, four bird, and one lizard species from carrion fly metabarcoding, all but one of which are known from BCI. Fly metabarcoding detected more mammal species than concurrent transect counts (29 sampling days, 13 species) and concurrent camera trapping (84 sampling days, 17 species), and detected 67% of the number of mammal species documented by 8 years of transect counts and camera trapping combined, although fly metabarcoding missed several abundant species. This study demonstrates that carrion fly metabarcoding is a powerful tool for mammal biodiversity surveys and has the potential to detect a broader range of species than more commonly used methods.     

Wood-inhabiting fungal communities in woody debris of Norway spruce (Picea abies (L.) Karst.), as reflected by sporocarps, mycelial isolations and T-RFLP identification

Wood-inhabiting fungi play a key role in forest ecosystems and constitute an essential part of forest biodiversity. We therefore examined the composition and abundance of wood-inhabiting fungi by three methods: sporocarp counts, mycelial culturing and direct amplification of internal transcribed spacer terminal restriction fragment length polymorphism from wood combined with sequencing of reference rDNA. Seven-year-old slash piles left after a thinning were analyzed in a 50-year-old Norway spruce plantation. Fifty-eight fungal species were detected from the piled branches and treetops. More species were revealed by sporocarp counts and cultured mycelia than by direct amplification from wood. In principle, sporocarp monitoring may reveal all fruiting taxa, but it poorly reflects their relative abundance in the wood. In contrast, terminal restriction fragment length polymorphism will record the most frequent fungal taxa in the wood, but it may overlook uncommon taxa. Culturing mycelia from wood gives a bias towards species favoured by the cultural medium. The results demonstrate the advantage and the limitations of these methods to be considered in analyses of fungal communities in wood.     

The importance of timing and number of surveys in fungal biodiversity research

Practical conservation of biological diversity is dependent on reliable knowledge about what kind, how much, and where the diversity is. To obtain such knowledge three questions, why, what, and how, must be answered before commencing any biodiversity survey. While the questions why and what are often value decisions and thus determined outside the realm of scientific research, the question about how the surveys are conducted lies in the heart of science. Here, we report an intensive repeated survey of wood-inhabiting fungi with the aim of determining the optimal timing and number of the surveys for reliable estimation of the diversity of this species group. The research focusing on the ecology of wood-inhabiting fungi has been increasing but little is known about the reliability of the methods. The variation in the estimates of diversity among surveys was high and the results varied between studied species groups. The site-scale detectability for species belonging to different groups varied from 10 to 95% depending on the survey month and the species group. We conclude that because detectability of many fungi turned out to be poor even when surveys were conducted at an optimal time, the common practice of using a single fruit body survey to estimate fungal diversity of any given area is not enough. We suggest that multiple surveys at an optimal time should be a norm in fungal diversity studies. Improper methodology results in unreliable outcomes that have potential to hamper our goal of conserving the biological diversity.