Death caps (Amanita phalloides) frequently establish from sexual spores, but individuals can grow large and live for more than a decade in invaded forests.

Global change is reshaping Earth's biodiversity, but the changing distributions of nonpathogenic fungi remain largely undocumented, as do mechanisms enabling invasions. The ectomycorrhizal Amanita phalloides is native to Europe and invasive in North America. Using population genetics and genomics, we sought to describe the life history traits of this successfully invading symbiotic fungus. To test whether death caps spread underground using hyphae, or aboveground using sexual spores, we mapped and genotyped mushrooms from European and US sites. Larger genetic individuals (genets) would suggest spread mediated by vegetative growth, while many small genets would suggest dispersal mediated by spores. To test whether genets are ephemeral or persistent, we also sampled from populations over time. At nearly every site and across all time points, mushrooms resolve into small genets. Individuals frequently establish from sexual spores. But at one Californian site, a single individual measuring nearly 10 m across dominated. At two Californian sites, the same genetic individuals were discovered in 2004, 2014, and 2015, suggesting single individuals (both large and small) can reproduce repeatedly over relatively long timescales. A flexible life history strategy combining both mycelial growth and spore dispersal appears to underpin the invasion of this deadly perennial ectomycorrhizal fungus.

Large and variable genome size unrelated to serpentine adaptation but supportive of cryptic sexuality in Cenococcum geophilum.

Estimations of genome size and its variation can provide valuable information regarding the genetic diversity of organisms and their adaptation potential to heterogeneous environments. We used flow cytometry to characterize the variation in genome size among 40 isolates of Cenococcum geophilum, an ectomycorrhizal fungus with a wide ecological and geographical distribution, obtained from two serpentine and two non-serpentine sites in Portugal. Besides determining the genome size and its intraspecies variation, we wanted to assess whether a relationship exists between genome size and the edaphic background of the C. geophilum isolates. Our results reveal C. geophilum to have one of the largest genome sizes so far measured in the Ascomycota, with a mean haploid genome size estimate of 0.208 pg (203 Mbp). However, no relationship was found between genome size and the edaphic background of the sampled isolates, indicating genetic and demographic processes to be more important for shaping the genome size variation in this species than environmental selection. The detection of variation in ploidy level among our isolates, including a single individual with both presumed haploid and diploid nuclei, provides supportive evidence for a possible cryptic sexual or parasexual cycle in C. geophilum (although other mechanisms may have caused this variation). The existence of such a cycle would have wide significance, explaining the high levels of genetic diversity and likelihood of recombination previously reported in this species, and adds to the increasing number of studies suggesting sexual cycles in previously assumed asexual fungi.

Invasive Californian death caps develop mushrooms unisexually and bisexually.

Canonical sexual reproduction among basidiomycete fungi involves the fusion of two haploid individuals of different mating types, resulting in a heterokaryotic mycelial body made up of genetically different nuclei. Using population genomics data and experiments, we discover mushrooms of the invasive and deadly Amanita phalloides can also be homokaryotic; evidence of sexual reproduction by single, unmated individuals. In California, genotypes of homokaryotic mushrooms are also found in heterokaryotic mushrooms, implying nuclei of homokaryotic mycelia are also involved in outcrossing. We find death cap mating is controlled by a single mating type locus, but the development of homokaryotic mushrooms appears to bypass mating type gene control. Ultimately, sporulation is enabled by nuclei able to reproduce alone as well as with others, and nuclei competent for both unisexuality and bisexuality have persisted in invaded habitats for at least 17 but potentially as long as 30 years. The diverse reproductive strategies of invasive death caps are likely facilitating its rapid spread, suggesting a profound similarity between plant, animal and fungal invasions.

Evidence of adaptive tolerance to nickel in isolates of Cenococcum geophilum from serpentine soils.

Selection for metal-tolerant ecotypes of ectomycorrhizal (ECM) fungi has been reported in instances of metal contamination of soils as a result of human activities. However, no study has yet provided evidence that natural metalliferous soils, such as serpentine soils, can drive the evolution of metal tolerance in ECM fungi. We examined in vitro Ni tolerance in isolates of Cenococcum geophilum from serpentine and non-serpentine soils to assess whether isolates from serpentine soils exhibited patterns consistent with adaptation to elevated levels of Ni, a typical feature of serpentine. A second objective was to investigate the relationship between Ni tolerance and specific growth rates (micro) among isolates to increase our understanding of possible tolerance/growth trade-offs. Isolates from both soil types were screened for Ni tolerance by measuring biomass production in liquid media with increasing Ni concentrations, so that the effective concentration of Ni inhibiting fungal growth by 50% (EC(50)) could be determined. Isolates of C. geophilum from serpentine soils exhibited significantly higher tolerance to Ni than non-serpentine isolates. The mean Ni EC(50) value for serpentine isolates (23.4 microg ml(-1)) was approximately seven times higher than the estimated value for non-serpentine isolates (3.38 microg ml(-1)). Although there was still a considerable variation in Ni sensitivity among the isolates, none of the serpentine isolates had EC(50) values for Ni within the range found for non-serpentine isolates. We found a negative correlation between EC(50) and micro values among isolates (r = -0.555). This trend, albeit only marginally significant (P = 0.06), indicates a potential trade-off between tolerance and growth, in agreement with selection against Ni tolerance in "normal" habitats. Overall, these results suggest that Ni tolerance arose among serpentine isolates of C. geophilum as an adaptive response to Ni exposure in serpentine soils.

In Colombia the Eurasian fungus Amanita muscaria is expanding its range into native, tropical Quercus humboldtii forests.

To meet a global demand for timber, tree plantations were established in South America during the first half of the 20th century. Extensive plantings of non-native species now are found in Brazil, Chile, Argentina, and Uruguay. In Colombia, miscellaneous plantations were established in the 1950s, during a period of intensive local logging, when policies to limit deforestation in native Quercus humboldtii forests were established. One unforeseen consequence of planting non-native trees was the simultaneous introduction and subsequent persistence of ectomycorrhizal fungi. We sought to document the origins and spread of the introduced Amanita muscaria found in Colombian plantations of the Mexican species Pinus patula, North American species P. taeda, and Australian species Acacia melanoxylon and Eucalyptus globulus. In Colombia, Amanita muscaria is establishing a novel association with native Q. humboldtii and has spread to local Q. humboldtii forests. According to a Bayesian phylogeny and haplotype analysis based on the nuclear rDNA internal transcribed spacer region ITS1-5.8-ITS2 (ITS barcode), A. muscaria individuals found in four exotic plant species, and those colonizing Q. humboldtii roots, have a Eurasian origin and belong to two Eurasian haplotypes. This is the first time the spread of an introduced mutualist fungus into native Colombian Q. humboldtii forests is reported. To arrest its spread, we suggest the use of local inocula made up of native fungi, instead of inocula of introduced fungi.

A Ni hyperaccumulator and a congeneric non-accumulator reveal equally effective defenses against herbivory.

The defense hypothesis is commonly used to explain the adaptive role of metal hyperaccumulation. We tested this hypothesis using two Brassicaceae congeneric species: Alyssum pintodasilvae, a Ni hyperaccumulator, and the non-accumulator Alyssum simplex both growing on serpentine soils in Portugal. Artificial diet disks amended with powdered leaves from each plant species were used to compare the performance (mortality, biomass change) and feeding behavior of Tribolium castaneum in no-choice and choice tests. The performance of T. castaneum was not affected at several concentrations of A. pintodasilvae or A. simplex in no-choice tests. However, the consumption of plant-amended disks was significantly lower than that of control disks, irrespectively of the species fed. Accordingly, when insects were given an alternative food choice, disks of both plant species were significantly less consumed than control disks. Moreover, insects did not discriminate between disks in the combination "A. pintodasilvae+A. simplex". Contrary to our expectations, these results suggest that both plant species have equally effective defenses against herbivory. While Ni is believed to be part of the deterrence mechanism in the hyperaccumulator A. pintodasilvae, it seems likely that organic compounds, possibly glucosinolates, play an important role in the defense of A. simplex or in both species.

Genetic diversity and differential in vitro responses to Ni in Cenococcum geophilum isolates from serpentine soils in Portugal.

Amplified fragment length polymorphism (AFLP) analysis was used to investigate the genetic diversity in isolates of the ectomycorrhizal fungus Cenococcum geophilum from serpentine and non-serpentine soils in Portugal. A high degree of genetic diversity was found among C. geophilum isolates; AFLP fingerprints showed that all the isolates were genetically distinct. We also assessed the in vitro Ni sensitivity in three serpentine isolates and one non-serpentine isolate. Only the non-serpentine isolate was significantly affected by the addition of Ni to the growth medium. At 30 microg g(-1) Ni, radial growth rate and biomass accumulation decreased to 73.3 and 71.6% of control, respectively, a highly significant inhibitory effect. Nickel at this concentration had no significant inhibitory effect on serpentine isolates, and so the fitness of serpentine isolates, as evaluated by radial growth rate and biomass yield, is likely unaffected by Ni in the field. In all isolates, the Ni concentration in the mycelia increased with increasing Ni concentration in the growth medium, but two profiles of Ni accumulation were identified. One serpentine isolate showed a linear trend of Ni accumulation. At the highest Ni exposure, the concentration of Ni in the mycelium of this isolate was in the hyperaccumulation range for Ni as defined for higher plants. In the remaining isolates, Ni accumulation was less pronounced and seems to approach a plateau at 30 microg g(-1) Ni. Because two profiles of Ni accumulation emerged among our Ni-insensitive serpentine isolates, this result suggests that different Ni detoxification pathways may be operating. The non-serpentine isolate whose growth was significantly affected by Ni was separated from the other isolates in the genetic analysis, suggesting a genetic basis for the Ni-sensitivity trait. This hypothesis is further supported by the fact that all isolates were maintained on medium without added Ni to avoid carry-over effects. However, because AFLP analysis failed to distinguish between serpentine and non-serpentine isolates, we cannot conclude that Ni insensitivity among our serpentine isolates is due to evolutionary adaptation. Screening a larger number of isolates, from different geographical origins and environments, should clarify the relationships between genetic diversity, morphology, and physiology in this important species.