Lethal amanitas (Amanita section Phalloideae) are a group of wild, fatal mushrooms causing many poisoning cases worldwide. However, the diversity and evolutionary history of these lethal mushrooms remain poorly known due to the limited sampling and insufficient gene fragments employed for phylogenetic analyses. In this study, five gene loci (nrLSU, ITS, rpb2, ef1-α and β-tubulin) with a widely geographic sampling from East and South Asia, Europe, North and Central America, South Africa and Australia were analysed with maximum-likelihood, maximum-parsimony and Bayesian inference methods. Biochemical analyses were also conducted with intention to detect amatoxins and phalloidin in 14 representative samples. Lethal amanitas were robustly supported to be a monophyletic group after excluding five species that were provisionally defined as lethal amanitas based on morphological studies. In lethal amanitas, 28 phylogenetic species were recognised by integrating molecular phylogenetic analyses with morphological studies, and 14 of them represented putatively new species. The biochemical analyses indicated a single origin of cyclic peptide toxins (amatoxins and phalloidin) within Amanita and suggested that this kind of toxins seemed to be a synapomorphy of lethal amanitas. Molecular dating through BEAST and biogeographic analyses with LAGRANGE and RASP indicated that lethal amanitas most likely originated in the Palaeotropics with the present crown group dated around 64.92 Mya in the early Paleocene, and the East Asia–eastern North America or Eurasia–North America–Central America disjunct distribution patterns were primarily established during the middle Oligocene to Miocene. The cryptic diversity found in this study indicates that the species diversity of lethal amanitas is strongly underestimated under the current taxonomy. The intercontinental sister species or sister groups relationships among East Asia and eastern North America or Eurasia–North America–Central America within lethal amanitas are best explained by the diversification model of Palaeotropical origin, dispersal via the Bering Land Bridge, followed by regional vicariance speciation resulting from climate change during the middle Oligocene to the present. These findings indicate the importance of both dispersal and vicariance in shaping the intercontinental distributions of these ectomycorrhizal fungi.

Mushroom poisoning is the main cause of mortality in food poisoning incidents in China. Although some responsible mushroom species have been identified, some were identified inaccuratly. This study investigated and analyzed 102 mushroom poisoning cases in southern China from 1994 to 2012, which involved 852 patients and 183 deaths, with an overall mortality of 21.48 %. The results showed that 85.3 % of poisoning cases occurred from June to September, and involved 16 species of poisonous mushroom: Amanita species (A. fuliginea, A. exitialis, A. subjunquillea var. alba, A. cf. pseudoporphyria, A. kotohiraensis, A. neoovoidea, A. gymnopus), Galerina sulciceps, Psilocybe samuiensis, Russula subnigricans, R. senecis, R. japonica, Chlorophyllum molybdites, Paxillus involutus, Leucocoprinus cepaestipes and Pulveroboletus ravenelii. Six species (A. subjunquillea var. alba, A. cf. pseudoporphyria, A. gymnopus, R. japonica, Psilocybe samuiensis and Paxillus involutus) are reported for the first time in poisoning reports from China. Psilocybe samuiensis is a newly recorded species in China. The genus Amanita was responsible for 70.49 % of fatalities; the main lethal species were A. fuliginea and A. exitialis. Russula subnigricans caused 24.59 % of fatalities, and five species showed mortality >20 % (A. fuliginea, A. exitialis, A. subjunquillea var. alba, R. subnigricans and Paxillus involutus). Mushroom poisoning symptoms were classified from among the reported clinical symptoms. Seven types of mushroom poisoning symptoms were identified for clinical diagnosis and treatment in China, including gastroenteritis, acute liver failure, acute renal failure, psychoneurological disorder, hemolysis, rhabdomyolysis and photosensitive dermatitis.

In the present work, loop-mediated isothermal amplification (LAMP) and hyperbranched rolling circle amplification (HRCA) methods were developed to detect and distinguish different lethal Amanita species. Specific LAMP primers and HRCA padlock probes for species-specific identification and a set of universal LAMP primers for lethal Amanita species were designed and tested. The results indicated that the LAMP-based assay was able to discriminate introclade lethal Amanita species but was not able to discriminate intraclade species perfectly, while the HRCA-based assay could discriminate whether introclade or intraclade species. The universal LAMP primers were positive for 10 lethal species of Amanita section Phalloideae and negative for 16 species of Amanita outside section Phalloideae. The detection limits of LMAP and HRCA were 10 and 1 pg of genomic DNA per reaction, respectively. In conclusion, the two methods could be rapid, specific, sensitive and low-cost tools for the identification of lethal Amanita species.