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.

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.

Investigating the allelic evolution of an imperfect microsatellite locus in the Hawaiian mushroom Rhodocollybia laulaha.

The evolutionary mechanisms that give rise to microsatellite alleles remain poorly understood in general and are especially understudied for fungal microsatellite loci. The unusual G28 microsatellite locus was developed from the Hawaiian mushroom Rhodocollybia laulaha. Here, we employ a novel approach to test for allele size homoplasy and examine competing mechanistic models of microsatellite evolution in the context of biogeographic expectations for this locus based on Hawaiian geologic history. Seven G28 alleles have been identified from a sampling of 153 individuals. The G28 locus is composed of a trinucleotide imperfect motif, which permits examination of the relationships between alleles and allows for detection of potential size homoplasy within the repetitive element. Alignment of G28 allele sequence data across multiple unrelated individuals suggests that alleles of like size are homologous within Hawaii. A variety of gap coding methods are explored in the inference of allele evolution. Length differences between alleles appear to be the result of polymerase slippage at multiple positions in the repetitive element, suggesting an intricate process of allelic evolution, which is not necessarily stepwise. Complex migration scenarios must be invoked to explain the current geographic distribution of alleles if their evolution was in fact sequential (from longest to shortest or from shortest to longest) as predicted by the "progression rule."

The response of ectomycorrhizal fungal inoculum to long-term increases in nitrogen supply.

The inoculum of ectomycorrhizal (EM) fungi was examined in a 16 y long nitrogen fertilization experiment maintained in a temperate oak savanna. To measure EM fungal inoculum, bur oak seedlings were grown in three types of bioassays: (i) intact soil cores that measure inoculum such as spores, mycelia and mycorrhizal roots; (ii) resistant propagule bioassays that measure inoculum types resistant to soil drying; and (iii) previously mycorrhizal root bioassays that measure the ability of EM fungi to colonize new roots from mycorrhizal roots. Colonization of bur oak seedlings was characterized by morphotyping and where necessary by restriction analysis and internal transcribed spacer (ITS) sequencing. Fourteen morphotypes were found in intact soil core bioassays with species of Cortinarius, Cenococcum and Russula abundant. Five morphotypes were found in resistant propagule bioassays with Cenococcum, a thelephoroid morphotype and a Wilcoxina-like ascomycete abundant and frequent. In intact soil core bioassays total percent root colonization and number of morphotypes were not affected by N supply in 2000 and 2001. However the composition of EM fungi colonizing oak seedling roots was different with increased N supply such that Russula spp. (primarily Russula aff. amoenolens) were most abundant at the highest level of N supply. Dominant Russula spp. did not colonize any roots in resistant propagule bioassays but did colonize oak seedling roots from previously mycorrhizal roots. Results suggest that in this savanna N supply can influence the kinds of inoculum propagules present and thereby might affect the dynamics of ectomycorrhizal communities by differentially influencing reproductive and colonization strategies.

Long-term increase in nitrogen supply alters above- and below-ground ectomycorrhizal communities and increases the dominance of Russula spp. in a temperate oak savanna.

• Here we examine the effects of increased nitrogen (N) supply on the ectomycorrhizal fungal communities of a temperate oak savanna. • In a 16-yr N-addition experiment in which replicate 1000 m2 plots received 0, 5.4 or 17 g N m-2  yr-1 , ectomycorrhizal sporocarp production was measured in the 14th, 15th and 16th year of fertilization. Ectomycorrhizal fungi (EMF) colonizing roots were examined by morphotyping-PCR-RFLP and sequence analysis in the 14th and 15th year of fertilization. • Total sporocarp richness was reduced by > 50% in both fertilization treatments in all 3 yrs, whereas Russula spp. produced approx. five times more sporocarps with 17 g N m-2  yr-1 . Below-ground, treatment-scale species richness and species area curves were lower with 17 g N m-2  yr-1 but richness, diversity indices and evenness at smaller spatial scales were not. Dominant fungi colonizing roots included Cenococcum geophilum, common in all treatments, Cortinarius spp., dominant in unfertilized plots, and Russula spp., dominant with 17 g N m-2  yr-1 . • Communities of EMF in this temperate deciduous ecosystem responded to N addition similarly to those of coniferous ecosystems in that increased N supply altered EMF diversity and community composition but differently in that dominance of Russula spp. increased.

Fungal communities influence root exudation rates in pine seedlings.

Root exudates are hypothesized to play a central role in belowground food webs, nutrient turnover, and soil C dynamics in forests, but little is known about the extent to which root-associated microbial communities influence exudation rates in trees. We used a novel experimental technique to inoculate loblolly pine (Pinus taeda L.) seedlings with indigenous forest fungi to examine how diverse fungal communities influence exudation. Surface-sterilized seeds were sown in intact, unsieved soil cores for 14 weeks to promote root colonization by fungi. After 14 weeks, we transferred seedlings and root-associated fungi into cuvettes and measured exudate accumulation in trap solutions. Both the abundance and identity of root-associated fungi influenced exudation. Exudation rates were greatest in root systems least colonized by ectomycorrhizal (ECM) fungi and most colonized by putative pathogenic and saprotrophic fungi. However, the ECM community composition was not a strong determinant of exudation rates. These results suggest that environmental conditions that influence the degree to which tree roots are colonized by pathogenic and saprotrophic vs. mutualistic fungi are likely to mediate fluxes of labile C in forest soils, with consequences for soil biogeochemistry and ecosystem processes.

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.

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.