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.

Genomes of lethal Amanita and Galerina mushrooms have gradually become available in the past ten years; in contrast the other known amanitin-producing genus, Lepiota, is still vacant in this aspect. A fatal mushroom poisoning case in China has led to acquisition of fresh L. venenata fruiting bodies, based on which a draft genome was obtained through PacBio and Illumina sequencing platforms. Toxin-biosynthetic MSDIN family and Porlyl oligopeptidase B (POPB) genes were mined from the genome and used for phylogenetic and statistical studies to gain insights into the evolution of the biosynthetic pathway. The analysis of the genome data illustrated that only one MSDIN, named LvAMA1, exits in the genome, along with a POPB gene. No POPA homolog was identified by direct homology searching, however, one additional POP gene, named LvPOPC, was cloned and the gene structure determined. Similar to ApAMA1 in A. phalloides and GmAMA1 in G. marginata, LvAMA1 directly encodes α-amanitin. The two toxin genes were mapped to the draft genome, and the structures analyzed. Furthermore, phylogenetic and statistical analyses were conducted to study the evolution history of the POPB genes. Compared to our previous report, the phylogenetic trees unambiguously showed that a monophyletic POPB lineage clearly conflicted with the species phylogeny. In contrast, phylogeny of POPA genes resembled the species phylogeny. Topology and divergence tests showed that the POPB lineage was robust and these genes exhibited significantly shorter genetic distances than those of the house-keeping rbp2, a characteristic feature of genes with horizontal gene transfer (HGT) background. Consistently, same scenario applied to the only MSDIN, LvAMA1, in the genome. To the best of our knowledge, this is the first reported genome of Lepiota. The analyses of the toxin genes indicate that the cyclic peptides are synthesized through a ribosomal mechanism. The toxin genes, LvAMA1 and LvPOPB, are not in the vicinity of each other. Phylogenetic and evolutionary studies suggest that HGT is the underlining cause for the occurrence of POPB and MSDIN in Amanita, Galerina and Lepiota, which are allocated in three distantly-related families.

The burgeoning accumulation of genomic data in recent years has revolutionized our understanding of fungal phylogenies and classifications. However, the genomic era also brings new challenges, as phylogenetic incongruences make the appearance of monophyly in some phylogenetic trees questionable. Existing criteria for constructing taxonomic systems, such as diagnostic characters and divergence time, become insufficient to address this challenge. Through order-level analyses of genomic data of the Subkingdom Dikarya within the Kingdom Fungi, we introduce the extended quadripartition internode certainty (EQP-IC) value as a novel criterion for constructing high-level fungal classifications, with a recommended threshold of 0.1 for each taxonomic rank. Suprageneric taxa with an EQP-IC value exceeding 0.1 exhibit reduced topological variation, suggesting a stronger correspondence with natural taxonomic category. This new criterion was also put into practice to investigate the derived suborders of mushroom-forming Agaricales, including three suborders, Agaricineae, Pluteineae, and Tricholomatineae (APT), that had been long-standing problems in phylogenetic analyses. In total, 142 genomes, including 64 newly generated ones, were utilized to reconstruct the phylogenetic relationships and delve into the phylogenetic incongruencies and evolutionary histories of APT. Our data suggested widespread and high-level incomplete lineage sorting (ILS) and introgression/hybridization (IH) present among suborders within the APT. Therefore, a dichotomous phylogenetic tree may not reflect the real relationships among the clades within the APT. Instead, their natural relationships may be reticulate. Three newly named suborders, Amanitineae, Macrocystidiineae, and Omphalinineae are added to the clade including APT. The new combination Baisuzhenia humphreyi, new genus Baisuzhenia, new family Baisuzheniaceae, and new suborder Baisuzheniineae are proposed to accommodate Stereopsis humphreyi, which shows an independent, but close relationship, with the clade formed by the six above-mentioned derived suborders of Agaricales.

Evolutionary radiation is widely recognized as a mode of species diversification, but the drivers of the rapid diversification of fungi remain largely unknown. Here, we used Amanitaceae, one of the most diverse families of macro-fungi, to investigate the mechanism underlying its diversification. The ancestral state of the nutritional modes was assessed based on phylogenies obtained from fragments of 36 single-copy genes and stable isotope analyses of carbon and nitrogen. Moreover, a number of time-, trait-, and paleotemperature-dependent models were employed to investigate if the acquisition of ectomycorrhizal (ECM) symbiosis and climate changes promoted the diversification of Amanitaceae. The results indicate that the evolution of ECM symbiosis has a single evolutionary origin in Amanitaceae. The earliest increase in diversification coincided with the acquisition of the ECM symbiosis with angiosperms in the middle Cretaceous. The recent explosive diversification was primarily triggered by the host-plant switches from angiosperms to the mixed forests dominated by Fagaceae, Salicaceae, and Pinaceae or to Pinaceae. Our study provides a good example of integrating phylogeny, nutritional mode evolution, and ecological analyses for deciphering the mechanisms underlying fungal evolutionary diversification. This study also provides new insights into how the transition to ECM symbiosis has driven the diversification of fungi.

Species of Amanita sect. Vaginatae (Fr.) Quél. are challenging to delimitate due to the morphological similarity or morphostasis among different taxa. In this study, a multi-locus (nuc rDNA region encompassing the internal transcribed spacers 1 and 2 with the 5.8S rDNA, the D1–D3 domains of nuc 28S rDNA, partial sequences of translation elongation factor 1-a, and the second largest subunit of RNA polymerase II) phylogeny was employed to investigate the species diversity of the section in eastern China. Sixteen species were recognized, including four new species; namely, A. circulata, A. multicingulata, A. orientalis, and A. sinofulva. They were documented with illustrated descriptions, ecological evidence, and comparisons with similar species. A key to the species of the section from eastern China is provided.

Species of Gymnopus sect. Levipedes are challenging to delimitate due to the morphological similarity among different taxa. In this study, morphological characteristics, molecular phylogenetic data, and ecological traits were integrated to investigate the species diversity of this section of southwestern China. A total of 17 species were documented in the region, including three new species, namely G. sinobrevipes, G. flavoalbus, G. yunnanensis, and two species new to the studied area—G. bicolor and G. ocior. Detailed descriptions and illustrations of the three new species are presented, along with comparisons to closely related or morphologically similar species. The remaining species included five that were originally described from southwestern China, four that were first identified from Europe, two from the Republic of Korea, and one from North America. Six of these species, originally described outside of China, currently lack molecular evidence to support their distributions in southwestern China. Finally, 11 species with morphological and molecular evidence were recognized in southwestern China. A key to these species is also provided.