Increased phylogenetic resolution within the ecologically important Rhizopogon subgenus Amylopogon using 10 anonymous nuclear loci.

Rhizopogon species are ecologically significant ectomycorrhizal fungi in conifer ecosystems. The importance of this system merits the development and utilization of a more robust set of molecular markers specifically designed to evaluate their evolutionary ecology. Anonymous nuclear loci (ANL) were developed for R. subgenus Amylopogon. Members of this subgenus occur throughout the United States and are exclusive fungal symbionts associated with Pterospora andromedea, a threatened mycoheterotrophic plant endemic to disjunct eastern and western regions of North America. Candidate ANL were developed from 454 shotgun pyrosequencing and assessed for positive amplification across targeted species, sequencing success, and recovery of phylogenetically informative sites. Ten ANL were successfully developed and were subsequently used to sequence representative taxa, herbaria holotype and paratype specimens in R. subgenus Amylopogon. Phylogenetic reconstructions were performed on individual and concatenated data sets by Bayesian inference and maximum likelihood methods. Phylogenetic analyses of these 10 ANL were compared with a phylogeny traditionally constructed using the universal fungal barcode nuc rDNA ITS1-5.8S-ITS2 region (ITS). The resulting ANL phylogeny was consistent with most of the species designations delineated by ITS. However, the ANL phylogeny provided much greater phylogenetic resolution, yielding new evidence for cryptic species within previously defined species of R. subgenus Amylopogon. Additionally, the rooted ANL phylogeny provided an alternate topology to the ITS phylogeny, which inferred a novel set of evolutionary relationships not identified in prior phylogenetic studies.

Genetic Analysis of the Aspergillus flavus Vegetative Compatibility Group to Which a Biological Control Agent That Limits Aflatoxin Contamination in U.S. Crops Belongs.

Some filamentous fungi in Aspergillus section Flavi produce carcinogenic secondary compounds called aflatoxins. Aflatoxin contamination is routinely managed in commercial agriculture with strains of Aspergillus flavus that do not produce aflatoxins. These non-aflatoxin-producing strains competitively exclude aflatoxin producers and reshape fungal communities so that strains with the aflatoxin-producing phenotype are less frequent. This study evaluated the genetic variation within naturally occurring atoxigenic A. flavus strains from the endemic vegetative compatibility group (VCG) YV36. AF36 is a strain of VCG YV36 and was the first fungus used in agriculture for aflatoxin management. Genetic analyses based on mating-type loci, 21 microsatellite loci, and a single nucleotide polymorphism (SNP) in the aflC gene were applied to a set of 237 YV36 isolates collected from 1990 through 2005 from desert legumes and untreated fields and from fields previously treated with AF36 across the southern United States. One haplotype dominated across time and space. No recombination with strains belonging to VCGs other than YV36 was detected. All YV36 isolates carried the SNP in aflC that prevents aflatoxin biosynthesis and the mat1-2 idiomorph at the mating-type locus. These results suggest that VCG YV36 has a clonal population structure maintained across both time and space. These results demonstrate the genetic stability of atoxigenic strains belonging to a broadly distributed endemic VCG in both untreated populations and populations where the short-term frequency of VCG YV36 has increased due to applications of a strain used to competitively exclude aflatoxin producers. This work supports the hypothesis that strains of this VCG are not involved in routine genetic exchange with aflatoxin-producing strains.

Microsatellite marker development for the coastal dune shrub Prunus maritima (Rosaceae).

UNLABELLED: • PREMISE OF THE STUDY: Microsatellite primers were developed in the beach plum, Prunus maritima, to investigate the genetic composition of remaining populations in need of conservation and, in future studies, to determine its relation to P. maritima var. gravesii. • METHODS AND RESULTS: Fourteen primer pairs were identified and tested in four populations throughout the species' geographic range. Of these 14 loci, 12 were shown to be polymorphic among a total of 60 P. maritima individuals sampled (15 individuals sampled from four populations). Among the polymorphic loci, the number of alleles ranged from two to 10 and observed heterozygosity of loci ranged from 0.07 to 0.93 among specimens tested. • CONCLUSIONS: These microsatellites will be useful in evaluating the population genetic composition of P. maritima and in developing approaches for further conservation and management of this species within the endangered coastal dune ecosystem of the northeastern United States.

Characterization of microsatellite markers for pinedrops, Pterospora andromedea (Ericaceae), from Illumina MiSeq sequencing.

PREMISE OF THE STUDY: Pterospora andromedea (Ericaceae) is a mycoheterotrophic plant endemic to North America with a disjunct distribution. Eastern populations are in decline compared to western populations. Microsatellite loci will allow comparison of genetic diversity in endangered to nonthreatened populations. • METHODS AND RESULTS: Illumina MiSeq sequencing resulted in development of 12 polymorphic microsatellite loci from 63 perfect microsatellite loci tested. One polymorphic locus was obtained from a traditional enrichment method. These 13 loci were screened across two western and two eastern populations. For western and eastern populations, respectively, number of alleles ranged from one to 10 and one to four, and observed heterozygosity ranged from 0.000 to 0.389 and 0.000 to 0.143. • CONCLUSIONS: These are the first microsatellite loci developed for Pterospora. They will be useful in conservation efforts of the eastern populations and for examination of population genetic parameters at different geographic scales and comparison with mycorrhizal fungal hosts.

Microsatellite primers for the fungi Rhizopogon kretzerae and R. salebrosus (Rhizopogonaceae) from 454 shotgun pyrosequencing.

PREMISE OF THE STUDY: Rhizopogon kretzerae and R. salebrosus (Rhizopogonaceae) are ectomycorrhizal fungi symbiotic with pines and the mycoheterotrophic plant Pterospora andromedea (Ericaceae). Microsatellite loci will allow population genetic study of fungal hosts to P. andromedea. • METHODS AND RESULTS: Shotgun pyrosequencing of R. kretzerae DNA resulted in primer development of 23 perfect microsatellite loci and screened across two populations each for R. kretzerae and R. salebrosus. Twelve loci were polymorphic in R. kretzerae populations, and 11 loci cross-amplified in R. salebrosus populations. For R. kretzerae and R. salebrosus, number of alleles was one to eight and one to nine, respectively, and observed heterozygosity ranged from 0.00-0.57 and 0.00-0.70, respectively. • CONCLUSIONS: These are the first microsatellite loci developed for any species within Rhizopogon subgenus Amylopogon. These microsatellite loci will be used in conservation genetic studies of rare to endangered eastern populations and to compare plant and fungal population genetic structure at different hierarchical levels.

Aspergillus flavus diversity on crops and in the environment can be exploited to reduce aflatoxin exposure and improve health.

Humans and animals are exposed to aflatoxins, toxic carcinogenic fungal metabolites, through consumption of contaminated food and feed. Aspergillus flavus, the primary causal agent of crop aflatoxin contamination, is composed of phenotypically and genotypically diverse vegetative compatibility groups (VCGs). Molecular data suggest that VCGs largely behave as clones with certain VCGs exhibiting niche preference. VCGs vary in aflatoxin-producing ability, ranging from highly aflatoxigenic to atoxigenic. The prevalence of individual VCGs is dictated by competition during growth and reproduction under variable biotic and abiotic conditions. Agronomic practices influence structures and average aflatoxin-producing potentials of A. flavus populations and, as a result, incidences and severities of crop contamination. Application of atoxigenic strains has successfully reduced crop aflatoxin contamination across large areas in the United States. This strategy uses components of the endemic diversity to alter structures of A. flavus populations and improve safety of food, feed, and the overall environment.

Intercontinental divergence in the Populus-associated ectomycorrhizal fungus, Tricholoma populinum.

The ectomycorrhizal fungus Tricholoma populinum is host-specific with Populus species. T. populinum has wind-dispersed progagules and may be capable of long-distance dispersal. In this study, we tested the hypothesis of a panmictic population between Scandinavia and North America. DNA sequences from five nuclear loci were used to assess phylogeographic structure and nucleotide divergence between continents. Tricholoma populinum was composed of Scandinavian and North American lineages with complete absence of shared haplotypes and only one shared nucleotide mutation. Divergence of these lineages was estimated at approx. 1.7-1.0 million yr ago (Ma), which occurred after the estimated divergence of host species Populus tremula and Populus balsamifera/Populus trichocarpa at 5 Ma. Phylogeographic structure was not observed within Scandinavian or North American lineages of T. populinum. Intercontinental divergence appears to have resulted from either allopatric isolation; a recent, rare long-distance dispersal founding event followed by genetic drift; or the response in an obligate mycorrhizal fungus with a narrow host range to contractions and expansion of host distribution during glacial and interglacial episodes within continents. Understanding present genetic variation in populations is important for predicting how obligate symbiotic fungi will adapt to present and future changing climatic conditions.

Suillus quiescens, a new species commonly found in the spore bank in California and Oregon.

Suillus quiescens sp. nov. is common under Pinus muricata on Santa Cruz and Santa Rosa Islands in the northern Channel Islands of California, and we subsequently found it fruiting at Point Reyes National Seashore on the central coast of California. Sequences from the internal transcribed spacer region show that it is distinct from all 44 species of Suillus tested, and features of its morphology separate it from all other unsequenced species. Suillus quiescens has a broader distribution than coastal California because it also was encountered as ectomycorrhizae on roots of pine seedlings from the eastern Sierra Nevada, coastal Oregon and the southern Cascade Mountains. The reason it had not been identified from these areas might be due to its resemblance to S. brevipes at maturity or it might be a rare fruiter that persists in the spore bank.

Inoculum potential of Rhizopogon spores increases with time over the first 4 yr of a 99-yr spore burial experiment.

In disturbed or pioneer settings, spores and sclerotia of ectomycorrhizal fungi serve as the necessary inoculum for establishment of ectomycorrhizal-dependent trees. Yet, little is known about the persistence of these propagules through time. Here, live field soil was inoculated with known quantities of basidiospores from four pine-associated species of Rhizopogon; these samples were then buried in retrievable containers, and pine seedling bioassays of serially diluted spore samples were used to measure spore viability. In the first 4 yr, no evidence of loss of spore viability was found in the four Rhizopogon species tested, but all four species exhibited dormancy in which a maximum of 1-8% of their spores were initially receptive to pine roots. There were some differences between species in overall inoculum potential of their spores, but all species broke dormancy at a statistically similar rate. This result provides evidence for spore dormancy in a common ectomycorrhizal genus, but it also precludes our ability to estimate the longevity of the spores accurately. Nevertheless these results, coupled with the observed patterns of Rhizopogon spore banks, suggest that at least decade-long durations are likely. As this experiment progresses, the true longevity of the spores will eventually be revealed.

Host islands within the California Northern Channel Islands create fine-scale genetic structure in two sympatric species of the symbiotic ectomycorrhizal fungus Rhizopogon.

We have examined fine-scale genetic structure of the symbiotic ectomycorrhizal fungi Rhizopogon occidentalis and R. vulgaris on two of the California Channel Islands using five and six microsatellite loci, respectively. Both Rhizopogon species are sympatric on Santa Cruz and Santa Rosa Islands and are ectomycorrhizal with bishop pine (Pinus muricata) on both islands or Santa Rosa Island Torrey pine (P. torreyana ssp. insularis) on Santa Rosa. The combination of disjunct pine host distributions and geographic barriers within and among the islands have created highly structured Rhizopogon populations over very short distances (8.5 km on Santa Cruz Island; F(ST) = 0.258, F(ST) = 0.056, R. occidentalis and R. vulgaris, respectively). Both species show similar patterns of genetic differentiation as a result of limited dispersal between host populations as revealed by a significant isolation by distance relationship (r = 0.69, P < 0.04; r = 0.93, P < 0.001, R. occidentalis and R. vulgaris, respectively) and Bayesian clustering analyses, and is most likely a function of the small foraging range of the few mammals that disperse Rhizopogon on these islands and the enormous spore bank characteristic of Rhizopogon species.

Biology of the ectomycorrhizal genus Rhizopogon. VI. Re-examination of infrageneric relationships inferred from phylogenetic analyses of ITS sequences.

Rhizopogon (Basidiomycota, Boletales) is a genus of hypogeous fungi that form ectomycorrhizal associations mostly with members of the Pinaceae. This genus comprises an estimated 100(+) species, with the greatest diversity found in coniferous forests of the Pacific northwestern United States. Maximum parsimony analyses of 54 nuclear ribosomal DNA internal transcribed spacer (ITS) sequences including 27 Rhizopogon and 10 Suillus species were conducted to test sectional relationships in Rhizopogon and examine phylogenetic relationships with the closely related epigeous genus, Suillus. Sequences from 10 Rhizopogon type collections were included in these analyses. Rhizopogon and Suillus were both monophyletic. Rhizopogon section Rhizopogon is not monophyletic and comprised two clades, one of which consisted of two well supported lineages characterized by several long insertions. Rhizopogon sections Amylopogon and Villosuli formed well supported groups, but certain species concepts within these sections were unresolved. Four species from section Fulviglebae formed a strongly supported clade within section Villosuli. Subgeneric taxonomic revisions are presented.