Peter G. Avis

A ‘dirty’ business: testing the limitations of terminal restriction fragment length polymorphism (TRFLP) analysis of soil fungi

Terminal restriction fragment length polymorphism (TRFLP) is an increasingly popular method in molecular ecology. However, several key limitations of this method have not been fully examined especially when used to study fungi. We investigated the impact of spore contamination, intracollection ribosomal DNA internal transcribed spacer (ITS) region variation, and conserved restriction enzyme recognition loci on the results produced by TRFLP to characterize soil fungal communities. We find that (i) the potential for nontarget structures such as spores to contribute DNA to target sample extractions is high; (ii) multiple fragments (i.e. ‘extra peaks’) per PCR primer‐restriction enzyme combination can be detected that are caused by restriction enzyme inefficiency and intracollection ribosomal DNA ITS variation; and (iii) restriction enzyme digestion in conserved vs. variable gene regions leads to different characterizations of community diversity. Based on these results, we suggest that studies employing TRFLP need to include information from known, identified fungi from sites within which studies take place and not to rely only on TRFLP profiles as a short cut to fungal community description.

Positive feedbacks between decomposition and soil nitrogen availability along fertility gradients

Background and aimsWe determined the relationship between site N supply and decomposition rates with respect to controls exerted by environment, litter chemistry, and fungal colonization.MethodsTwo reciprocal transplant decomposition experiments were established, one in each of two long-term experiments in oak woodlands in Minnesota, USA: a fire frequency/vegetation gradient, along which soil N availability varies markedly, and a long-term N fertilization experiment. Both experiments used native Quercus ellipsoidalis E.J. Hill and Andropogon gerardii Vitman leaf litter and either root litter or wooden dowels.ResultsLeaf litter decay rates generally increased with soil N availability in both experiments while belowground litter decayed more slowly with increasing soil N. Litter chemistry differed among litter types, and these differences had significant effects on belowground (but not aboveground) decay rates and on aboveground litter N dynamics during decomposition. Fungal colonization of detritus was positively correlated with soil fertility and decay rates.ConclusionsHigher soil fertility associated with low fire frequency was associated with greater leaf litter production, higher rates of fungal colonization of detritus, more rapid leaf litter decomposition rates, and greater N release in the root litter, all of which likely enhance soil fertility. During decomposition, both greater mass loss and litter N release provide mechanisms through which the plant and decomposer communities provide positive feedbacks to soil fertility as ultimately driven by decreasing fire frequency in N-limited soils and vice versa.

Pooled samples bias fungal community descriptions

We tested the accuracy of molecular analyses for recovering the species richness and structure of pooled fungal communities of known composition. We constructed replicate pools of 2–20 species and analysed these pools by two separate pooling‐DNA extraction procedures and three different molecular analyses (Automated Ribosomal Intergenic Spacer Analysis (ARISA), terminal restriction fragment length polymorphism (T‐RFLP) and clone library‐sequencing). None of the methods correctly described the known communities. Only clone library‐sequencing with high sequencing per pool (∼100 clones) recovered reasonable estimates of richness. Frequency data were skewed with all procedures and analyses. These results indicate that the error introduced by pooling samples is significant and problematic for ecological studies of fungal communities.

Good-Enough RFLP Matcher (GERM) program

A spreadsheet-based program (Good-Enough RFLP Matcher or GERM) is presented that matches unknown restriction fragment length polymorphism (RFLP) patterns of ectomycorrhizal fungi to a database of known ectomycorrhizal fungi. The program uses three simple methods to determine whether a sample matches a known: (1) Forward Matching: whether every band in the unknown is present in a known sample within a given error range; (2) Backward Matching: whether every band in the known sample is present in the unknown within a given error range; (3) Sum of Bands: whether the sum of all bands in the known and unknown are similar within a given error range. The program is available through the web page of this journal.

Ectomycorrhizal fungal communities of oak savanna are distinct from forest communities

Oak savanna is one of the most endangered ecosystems of North America, with less than 0.02% of its original area remaining. Here we test whether oak savanna supports a unique community of ectomycorrhizal fungi, a higher diversity of ectomycorrhizal fungi or a greater proportional abundance of ascomycete fungi compared with adjacent areas where the absence of fire has resulted in oak savanna conversion to oak forest. The overall fungal community was highly diverse and dominated by Cenococcum geophilum and other ascomycetes, Cortinarius, Russula, Lactarius and Thelephoraceae. Oak savanna mycorrhizal communities were distinct from oak forest communities both aboveground (sporocarp surveys) and belowground (RFLP identification of ectomycorrhizal root tips); however total diversity was not higher in oak savanna than oak forests and there was no evidence of a greater abundance of ascomycetes. Despite not having a higher local diversity than oak forests, the presence of a unique fungal community indicates that oak savanna plays an important role in maintaining regional ectomycorrhizal diversity.

Ectomycorrhizal iconoclasts: the ITS rDNA diversity and nitrophilic tendencies of fetid Russula

Fetid Russula are frequently dominant ectomycorrhizal fungi, and some appear to be especially nitrophilic. However, little is known about their phylogenetic relationships or how common nitrophilic traits are in this group. This study addresses this gap and presents a phylogenetic analysis of ITS rDNA sequences and a meta-analysis of studies that examine ectomycorrhizal fungi response to nitrogen increase. The phylogenetic analysis indicates that (i) this lineage contains numerous unidentified taxa; (ii) the taxa have distinct geographic distributions; and (iii) the misuse of names such as R amoenolens, R. foetens or R. pectinatoides is common. Twenty-three well supported phylotypes were identified and include clades specific to western North America, eastern North America, Europe and Asia, while morphologically similar collections from tropical-equatorial regions are distinct. The metaanalysis shows that nitrophilic tendencies appear throughout fetid Russulas suggesting that this character is not an isolated trait within this subgenus but instead is a more general feature of the group overall. Mapping these tendencies across a broader portion of the Russulaceae shows that this trait is more regularly found in the basal Russula lineages and Lactarius spp., suggesting that this ability evolved early in these fungi.

Variability in the IGS1 region of Rhodocollybia laulaha: is it allelic, genomic, or artificial?

We tested the nuclear ribosomal intergenic spacer 1 (IGS1) to evaluate: 1) the utility of this gene region in the study of intra-lineage variation in the Hawaiian mushroom Rhodocollybia laulaha (Basidiomycota, Marasmiaceae); 2) the possibility of PCR-mediated artifacts from multiple cloned IGS1 inserts for five of the nine specimens included; and 3) the assignment of individual specimens to specific haplotype group genotypes. The IGS1 was sequenced for nine specimens of R. laulaha and multiple cloned copies of the IGS1 were sequenced for five of those nine to compare intra-specimen variation (i.e. among paralogous copies in a single genome) and inter-specimen variation (i.e. among different specimens). Three haplotype groups were identified with 25 of 510 aligned base pairs being variable (17 substitutions and eight indels). The observed variation could be due to the presence of various alleles of the IGS1 locus and/or variation among paralogous copies within a single genome. Issues associated with PCR error including artificial recombination and/or Taq error and the potential inefficiency of concerted evolution are considered as possible agents of variation.

Analysis of morphological, ecological and molecular characters of Russula pectinatoides Peck and Russula praetervisa Sarnari, with a description of the new taxon Russula recondita Melera & Ostellari

Fetid Russulas belong to a homogeneous group including species with an unclear taxonomic classification. In particular, the correct interpretation of Russula pectinatoides Peck has remained unresolved, due to the reference collection deposited by C. H. Peck in 1906 consisting of poorly described specimens which were morphologically attributed to at least two different species by subsequent mycologists. Another species considered emblematic is R. praetervisa Sarnari, because it is closely related to R. pectinatoides, from which it was separated on the basis of the morphology of European samples found in Mediterranean habitats. In this paper, we review the interpretation of R. pectinatoides and R. praetervisa according to different authors, and we discuss the taxonomic status of these taxa in the light of molecular analyses carried out on Peck’s reference specimens (n = 3) and 181 samples archived in European and American museums. Sequences of ITS1-5.8S ribosomal RNA-ITS2 regions indicated that R. pectinatoides reference samples deposited by Peck consisted of at least three different species. R. praetervisa was supported as an independent taxon, as it was molecularly distinct from other fetid Russulas. The analysis of museum samples revealed the necessity to describe the species to which in Europe the name R. pectinatoides is commonly applied as a new species, named R. recondita Melera & Ostellari.

Testing the “one-log-one-genet” hypothesis: methodological challenges of population sampling for the Hawaiian wood-decay fungus Rhodocollybia laulaha

We test our “one-log-one-genet” sampling method for the Hawaiian mushroom Rhodocollybia laulaha that posits all R. laulaha mushrooms collected from a single log represent a single genet. We also examine the potential expansion of single genets beyond the confines of one log and the temporal persistence of genets in nature. Finally, we estimate error rates in AFLP scoring. To our knowledge, this is one of few examinations of naturally occurring fungal genets in the tropics and a novel report of AFLP error rates in fungi. Forty-six mushrooms from seven logs were genotyped with the IGS1 locus, two microsatellite loci and 184 AFLP loci from three primer pair combinations. One hundred fifty-three mushroom collections representing the geographic range of R. laulaha were genotyped with the IGS1 and microsatellite loci. The probabilities of two genets sharing identical multilocus genotypes by chance (without actually being the same genet) were calculated for each genotype recovered. The data suggest that R. laulaha mushrooms from one log typically represent one genet, that genets might expand beyond the confines of a single log and that a single genet may persist in a collecting site for as much as 13 y. We offer initial evidence to support the “one-log-one genet” sampling method and the idea that R. laulaha vegetative expansion and persistence in nature might be common. In addition, we caution against exclusive use of AFLP loci for identifying fungal genets due to relatively high error rates in scoring.

An Intact Soil Core Bioassay for Cultivating Forest Ectomycorrhizal Fungal Communities

Ectomycorrhizal and other root-associated fungi play a critical role in forests by influencing plant community structure and nutrient dynamics; however, given that most fungal taxa are difficult to cultivate, methods for cultivating natural fungal communities are needed to better understand the ecological consequences of species gains and losses. We grew loblolly pine (Pinus taeda) seedlings in intact, undisturbed soil cores collected from the Duke Forest (North Carolina, USA) and used molecular tools to measure how the fungal communities that colonized the seedlings resembled those reported in previous field-based investigations. Overall, we found substantial overlap between fungi, especially ectomycorrhizal fungi, recovered with our method and those from in situ studies, with over 70% of the sequences and nearly 50% of the OTU in common. This study shows that the intact core ‘baiting’ method greatly facilitates the study of natural fungal communities composited by ectomycorrhizal and other fungi, is a significant improvement over bioassays that homogenize soils, and can be used to experimentally study functioning of ecologically-relevant fungal communities of forest ecosystems.