Genes and evolutionary fates of the amanitin biosynthesis pathway in poisonous mushrooms.

The deadly toxin α-amanitin is a bicyclic octapeptide biosynthesized on ribosomes. A phylogenetically disjunct group of mushrooms in Agaricales (Amanita, Lepiota, and Galerina) synthesizes α-amanitin. This distribution of the toxin biosynthetic pathway is possibly related to the horizontal transfer of metabolic gene clusters among taxonomically unrelated mushrooms with overlapping habitats. Here, our work confirms that two biosynthetic genes, P450-29 and FMO1, are oxygenases important for amanitin biosynthesis. Phylogenetic and genetic analyses of these genes strongly support their origin through horizontal transfer, as is the case for the previously characterized biosynthetic genes MSDIN and POPB. Our analysis of multiple genomes showed that the evolution of the α-amanitin biosynthetic pathways in the poisonous agarics in the Amanita, Lepiota, and Galerina clades entailed distinct evolutionary pathways including gene family expansion, biosynthetic genes, and genomic rearrangements. Unrelated poisonous fungi produce the same deadly amanitin toxins using variations of the same pathway. Furthermore, the evolution of the amanitin biosynthetic pathway(s) in Amanita species generates a much wider range of toxic cyclic peptides. The results reported here expand our understanding of the genetics, diversity, and evolution of the toxin biosynthetic pathway in fungi.

Pulvinic Acid Derivative Pigments in Lanmaoa asiatica and L. macrocarpa.

Lanmaoa asiatica G. Wu & Zhu L. Yang and L. macrocarpa N. K. Zeng, H. Chai & S. Jiang are two important gourmet bolete in China, and locally named "Jian Shou Qing" meaning their fruiting bodies turn blue after bruising. The genus represents a distinct lineage in Boletaceae. The pigment(s) associated with the discoloration in Lanmaoa has not been identified. The aim of this study was to determine the pigment(s) underpinning the bluing reaction of L. asiatica and L. macrocarpa when bruised. Potential compounds were isolated by HPLC and identified by LC-HRMS and NMR. In total five to six pigments of hydroxylated pulvinic acid derivatives were detected with similar distribution patterns in both L. asiatica and L. macrocarpa, which by abundance were variegatic acid, variegatorubin, xerocomic acid (and/or isoxerocomic acid), xerocomorubin, and atromentic acid. Variegatic acid, the most abundant pigment, was isolated by HPLC, and the structure was further characterized by NMR. The amount of variegatic acid increased after regular cooking, which may suggest its enhanced health benefit as human diet. The types of pigments that cause bluing reactions often differ among families of Boletales. Our results showed that the pigments in Lanmaoa belong to the category of hydroxylated pulvinic acid derivatives, the major bluing compounds in Boletaceae.

Historical biogeography and diversification of ringless Amanita (section Vaginatae) support an African origin and suggest niche conservatism in the Americas.

Ectomycorrhizal fungi (ECM) sustain nutrient recycling in most terrestrial ecosystems, yet we know little about what major biogeographical events gave rise to present-day diversity and distribution patterns. Given the strict relationship between some ECM lineages and their hosts, geographically well-sampled phylogenies are central to understanding major evolutionary processes of fungal biodiversity patterns. Here, we focus on Amanita sect. Vaginatae to address global diversity and distribution patterns. Ancestral-state-reconstruction based on a 4-gene timetree with over 200 species supports an African origin between the late Paleocene and the early Eocene (ca. 56 Ma). Major biogeographic "out-of-Africa" events include multiple dispersal events to Southeast Asia (ca. 45-21 Ma), Madagascar (ca. 18 Ma), and the current Amazonian basin (ca. 45-36 Ma), the last two likely trans-oceanic. Later events originating in Southeast Asia involve Nearctic dispersal to North America (ca. 20-5 Ma), Oceania (Australia and New Zealand; ca. 15 Ma), and Europe (ca. 10-5 Ma). Subsequent dispersals were also inferred from Southeast Asia to East Asia (ca. 4 Ma); from North America to East Asia (ca. 11-8 Ma), Southeast Asia (ca. 19-2 Ma), Northern Andes (ca. 15 Ma), and Europe (ca. 15-2 Ma), respectively; and from the Amazon to the Caribbean region (ca. 25-20 Ma). Finally, we detected a significant increase in the net diversification rates in the branch leading to most northern temperate species in addition to higher state-dependent diversification rates in temperate lineages, consistent with previous findings. These results suggest that species of sect. Vaginatae likely have higher dispersal ability and higher adaptability to new environments, in particular compared to those of its sister clade, sect. Caesareae. Overall, the much wider distribution of A. sect. Vaginatae, from pan-tropical to pan-arctic, provides a unique window to understanding niche conservatism across a species-rich clade of ECM fungi.

Genome of lethal Lepiota venenata and insights into the evolution of toxin-biosynthetic genes.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Genetic diversity and structure of core collection of winter mushroom (Flammulina velutipes) developed by genomic SSR markers.

BACKGROUND: A core collection is a subset of an entire collection that represents as much of the genetic diversity of the entire collection as possible. The establishment of a core collection for crops is practical for efficient management and use of germplasm. However, the establishment of a core collection of mushrooms is still in its infancy, and no established core collection of the economically important species Flammulina velutipes has been reported. RESULTS: We established the first core collection of F. velutipes, containing 32 strains based on 81 genetically different F. veltuipes strains. The allele retention proportion of the core collection for the entire collection was 100%. Moreover, the genetic diversity parameters (the effective number of alleles, Nei's expected heterozygosity, the number of observed heterozygosity, and Shannon's information index) of the core collection showed no significant differences from the entire collection (p > 0.01). Thus, the core collection is representative of the genetic diversity of the entire collection. Genetic structure analyses of the core collection revealed that the 32 strains could be clustered into 6 groups, among which groups 1 to 3 were cultivars and groups 4 to 6 were wild strains. The wild strains from different locations harbor their own specific alleles, and were clustered stringently in accordance with their geographic origins. Genetic diversity analyses of the core collection revealed that the wild strains possessed greater genetic diversity than the cultivars. CONCLUSION: We established the first core collection of F. velutipes in China, which is an important platform for efficient breeding of this mushroom in the future. In addition, the wild strains in the core collection possess favorable agronomic characters and produce unique bioactive compounds, adding value to the platform. More attention should be paid to wild strains in further strain breeding.

The genus Pulveroboletus (Boletaceae, Boletales) in China.

Species of Pulveroboletus (Boletaceae, Boletales) in China are investigated on the basis of morphology and molecular phylogenetic analyses of DNA sequences from nuc rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S rDNA (ITS) and nuc 28S rDNA D1-D2 domains (28S) and sequences from the translation elongation factor 1-α gene (TEF1). Nine species are recognized in the country, three of them are described as new: P. flaviscabrosus, P. rubroscabrosus, and P. subrufus, five of them are previous described taxa: P. brunneopunctatus, P. brunneoscabrosus, P. macrosporus, P. reticulopileus, and P. sinensis, the remaining one is tentatively named P. cf. ridleyi, which will be further studied in the future. A key to the Chinese taxa of the genus is provided. Unexpectedly, the current study did not identify disjunct populations of Pulveroboletus species in East Asia and North/Central America.

Ecological and physical barriers shape genetic structure of the Alpine porcini (Boletus reticuloceps).

The Alpine porcini, Boletus reticuloceps, is an ectomycorrhizal mushroom distributed in subalpine areas of Southwest China, central China, and Taiwan Island. This distribution pattern makes it an ideal organism to infer how ectomycorrhizal fungi have reacted to historical tectonic and climatic changes, and to illustrate the mechanism for the disjunction of organisms between Southwest China and Taiwan. In this study, we explored the phylogeographic pattern of B. reticuloceps by microsatellite genotyping, DNA sequencing, ecological factor analysis, and species distribution modeling. Three genetic groups from the East Himalayas (EH), northern Hengduan Mountains (NHM), and southern Hengduan Mountains (SHM), were identified. The earlier divergent SHM group is found under Abies in moister environments, whereas the EH and NHM groups, which are physically separated by the Mekong-Salween Divide, are found mainly under Picea in drier environments. Samples from Taiwan showed a close relationship with the SHM group. High mountains did not form dispersal barriers among populations in each of the EH, NHM, and SHM groups, probably due to the relatively weak host specificity of B. reticuloceps. Our study indicated that ecological heterogeneity could have contributed to the divergence between the SHM and the NHM-EH groups, while physical barriers could have led to the divergence of the NHM and the EH groups. Dispersal into Taiwan via Central China during the Quaternary glaciations is likely to have shaped its disjunct distribution.

Diversity and distribution patterns of root-associated fungi on herbaceous plants in alpine meadows of southwestern China.

The diversity of root-associated fungi associated with four ectomycorrhizal herbaceous species, Kobresia capillifolia, Carex parva, Polygonum macrophyllum and Potentilla fallens, collected in three sites of alpine meadows in southwestern China, was estimated based on internal transcribed spacer (ITS) rDNA sequence analysis of root tips. Three hundred seventy-seven fungal sequences sorted to 154 operational taxonomical units (sequence similarity of ≥ 97% across the ITS) were obtained from the four plant species across all three sites. Similar taxa (in GenBank with ≥ 97% similarity) were not found in GenBank and/or UNITE for most of the OTUs. Ectomycorrhiz a made up 64% of the fungi operational taxonomic units (OTUs), endophytes constituted 4% and the other 33% were unidentified root-associated fungi. Fungal OTUs were represented by 57% basidiomycetes and 43% ascomycetes. Inocybe, Tomentella/Thelophora, Sebacina, Hebeloma, Pezizomycotina, Cenococcum geophilum complex, Cortinarius, Lactarius and Helotiales were OTU-rich fungal lineages. Across the sites and host species the root-associated fungal communities generally exhibited low host and site specificity but high host and sampling site preference. Collectively our study revealed noteworthy diversity and endemism of root-associated fungi of alpine plants in this global biodiversity hotspot.

Three new species of Physalacria from China, with a key to the Asian taxa.

Three new and one previously described species of Physalacria (Physalacriaceae, Agaricales) are reported from China. Specimens of two additional species described from Malaysia and North America were studied for comparison. Placements of these species were corroborated based on morphological observations and molecular evidence from partial sequences of the nuc rDNA internal transcribed spacer regions (ITS) and the 28S D1-D3 region, and genes for translation elongation factor 1-α (tef1α) and the second largest subunit of RNA polymerase II (rpb2). These new species of Physalacria distributed in subtropical China were found on rotten wood of broadleaf trees or bamboo and possess stipitate-capitate basidiomata with four-spored basidia, clamp connections and smooth, inamyloid basidiospores. To facilitate studies of the genus in Asia, a key is provided for all Physalacria species reported from this region.

Lethal Amanita species in China.

Lethal amanitas (Amanita sect. Phalloideae) cause many casualties worldwide. Recent molecular phylogenetic studies revealed diverse lethal Amanita spp. in China. Here a 5-gene phylogeny (nuc rDNA region encompassing the internal transcribed spacers 1 and 2 with the 5.8S rDNA, the D1-D3 domains of nuc 28S rDNA, and partial RNA polymerase II second largest subunit, translation elongation factor 1-α and ß-tubulin genes) is used to investigate the phylogenetic lineages and species delimitation in this section. Thirteen species are recognized, including four new species, namely A. griseorosea, A. molliuscula, A. parviexitialis, and A. subfuliginea They are documented with morphological, multigene phylogenetic, and ecological evidence, line drawings, and photographs and compared with similar species. A key to the Chinese lethal Amanita species is provided.

The genus Retiboletus in China.

Species of the genus Retiboletus (Boletaceae, Boletales) in China are investigated based on morphology and phylogenetic analyses of DNA sequences from nuc rDNA internal transcribed spacer (ITS) and partial 28S regions and sequences from the translation elongation factor 1-a gene (tef1a). Six lineages are recovered among the collections studied. Five of these are documented and presented in the present paper, including three new species and two new combinations. The remaining species is not described due to the paucity of material. The specimens from China identified as "R. ornatipes" or "R. retipes" are in fact R. sinensis or R. kauffmanii, those labeled "R. griseus" are either R. fuscus or R. pseudogriseus A key to all known taxa of the genus is provided. Phylogenetic relationships of taxa within Retiboletus are partially resolved. A preliminary biogeographical analysis shows that allied species of Retiboletus between eastern Asia and North/Central America are common but there are no Retiboletus species common to both continents. Species of Retiboletus in Japan and southern China are conspecific or closely related.

Four new species in Leucoagaricus (Agaricaceae, Basidiomycota) from Asia.

The genus Leucoagaricus has been well studied in Europe. However, species diversity of Leucoagaricus in Asia remains poorly known, especially in the mountains of southwestern China, a hot spot for biodiversity. Based on morphological characters and molecular data, four new species are described, La. asiaticus, La. subcrystallifer, La. subpurpureolilacinus and La. truncatus. Detailed morphological descriptions, drawings of microstructures for novel taxa and comparisons with closely allied taxa are provided. Phylogenetic analyses inferred from internal transcribed spacer (ITS) and region 6-7 of the gene for the second largest subunit of RNA polymerase II (rpb2) sequences show that the novel taxa are nested within a well-supported clade jointly formed by members of Leucoagaricus section Rubrotincti and subgenus Sericeomyces.

Multi-locus phylogeny of lethal amanitas: implications for species diversity and historical biogeography.

BACKGROUND: 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 ß-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. RESULT: 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. CONCLUSION: 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.

Molecular characterization of pHRDV1, a new virus-like mobile genetic element closely related to pleomorphic viruses in haloarchaea.

A novel haloarchaeal plasmid, pHRDV1 (13,053 bp), was isolated from the haloarchaeal isolate Halorubrum sp. T3. Molecular and bioinformatics analyses showed that this element is a double-stranded circular DNA molecule containing two putative transcripts with opposite directions. The amino acid sequences of six of the nineteen predicted open reading frames were similar to those found in haloarchaeal pleomorphic viruses, such as Halorubrum pleomorphic virus 3 and Halogeometricum pleomorphic virus 1. There was also a strong conservation in gene order between the plasmid and these viruses. All three conserved viral proteins (VPs), which are characteristic of haloarchaeal pleomorphic viruses VP3, VP4 and VP8, were found in pHRDV1. Furthermore, a typical repressor-operator system similar to haloarchaeal myovirus φCh1, was found on the genome of pHRDV1. However, no viral particles were detected in the supernatants of Halorubrum sp. T3, either in the presence or absence of mitomycin C. These results imply that plasmid pHRDV1 is a distinctive virus-like mobile genetic element that harbors some unique properties that make it different from all of the known haloarchaeal plasmids or viruses.

Molecular phylogeny and taxonomy of the genus Veloporphyrellus.

Veloporphyrellus is a genus known from North and Central America, southeastern Asia, and Africa. Because species of this genus are phenotypically similar to some taxa in several genera, such as Boletellus, Leccinum, Strobilomyces, Suillus and Tylopilus s.l. belonging to Boletales, its phylogenetic disposition has never been addressed. We analyzed four DNA regions, the nuclear ribosomal LSU and tef-1α, and the mitochondrial mtSSU and atp6 genes, to investigate the phylogenetic disposition of Veloporphyrellus. Although the monophyly of the genus and its systematic placement within the Boletaceae was well supported, its relationship to other genera was not resolved. Morphologically Veloporphyrellus is distinguished from other boletoid genera by the combination of the pinkish or grayish pink hymenophore, the membranous veil hanging on the pilea margin, the trichoderm-like pileus covering and the smooth basidiospores. Five species, including two new species and two new combinations, are described and illustrated. A key to the species of Veloporphyrellus also is provided.

The taxonomic foundation, species circumscription and continental endemisms of Singerocybe: evidence from morphological and molecular data.

The genus Singerocybe (Tricholomataceae, Agaricales, Basidiomycota) has been the subject of controversy since its proposal in 1988. Its taxonomic foundation, species circumscription and geographical distribution have not yet been examined with molecular sequence data. In this study phylogenetic analyses on this group of fungi were conducted based on collections from Europe, eastern Asia, southern Asia, North America and Australia, with four nuclear markers, ITS, nrLSU, tef1-α and rpb2. Molecular phylogenetic analyses, together with morphological observations, strongly support Singerocybe as a monophyletic group and identify the vesicles in the pileal and stipe cuticle as a synapomorphy of this genus. Seven species are recognized in the genus, including one new species and four new combinations. Clitocybe trogioides and Clitocybe trogioides var. odorifera are synonyms of Singerocybe humilis and Singerocybe alboinfundibuliformis respectively. Most of these species are geographically restricted in their distributions. Furthermore our study expands the distribution range of Singerocybe from the North Temperate Zone to Australia (Tasmania) and tropical southern Asia.

Biogeography and community assembly of soil fungi from alpine meadows in southwestern China show the importance of climatic selection.

Soil fungi are pivotal in alpine and arctic ecosystems that are vulnerable to climate changes. Previous studies have shown broad connections between soil fungi in the arctic and alpine regions, but most of these studies are mainly from Europe and North America, with more sporadic studies from East Asia. Currently, little is known about the biogeographic relationships between soil fungi in alpine meadows of southwestern China (AMSC) and other regions of the world. In addition, the regional-scale spatial patterns of fungal communities in the AMSC, as well as their driving factors and ecological processes, are also poorly understood. In this study, we collected roots and surrounding soils of two dominant ectomycorrhizal plants, Bistorta vivipara and B. macrophylla from the AMSC, and performed bioinformatic and statistical analyses based on high-throughput sequencing of ITS2 amplicons. We found that: (1) fungi from the AMSC were closely related with those from boreal forests and tundra, and saprotrophic fungi had higher dispersal potential than ectomycorrhizal fungi; (2) community compositions exhibited clear divergences among geographic regions and between root and soil samples; (3) climate was the predominant factor driving regional-scale spatial patterns but had less explanatory power for saprotrophic and total fungi from roots than those from soils; (4) homogeneous selection and drift were the key ecological processes governing community assembly, but in communities of saprotrophic and total fungi from soil samples, drift contributed less and its role was partially replaced by dispersal limitation. This study highlights the importance of climatic selection and stochastic processes on fungal community assembly in alpine regions, and emphasizes the significance of simultaneously investigating fungi with different trophic modes and from both roots and soils.

A contribution to the knowledge of the genus Infundibulicybe (Tricholomatineae, Agaricales) in China: Two new species and five redescribed taxa.

In China, species of the genus Infundibulicybe are often confused with other taxa and misdetermined. Here we describe two newly discovered species, I. bispora and I. ellipsospora, and redescribe five known taxa of this genus present in China. These identifications are supported by both morphological and DNA-based phylogenetic evidence. Additionally, a key to all known species of Infundibulicybe is provided.

Three new species and a new record of Cortinarius section Camphorati from southwestern China.

Cortinarius sect. Camphorati consists of telamonioid species with violet basidioma, strong odor, and distinct cheilocystidia. In this study, phylogenetic analysis based on nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and partial nuc rDNA 28S sequences has revealed three new species of the section from southwestern China, namely, C. longicystidiatus, C. megacystidiatus, and C. paraputorius, and a newly recorded species from southwestern China, viz., C. camphoratus, supplemented by the support from calculations of genetic distances of ITS sequences. Detailed descriptions of the four species are given with photographs, and their micromorphological characteristics are statistically compared. For species delimitation within the section, the size of basidiospores, the morphology of cheilocystidia, and the associated vegetation types and tree species are informative. A key to species in section Camphorati from the Northern Hemisphere is provided, and their geographic distributions and ecology are discussed.

Mushrooms Adapted to Seawater: Two New Species of Candolleomyces (Basidiomycota, Agaricales) from China.

Marine fungi have been studied for a long history in many realms, but there are few reports on marine mushrooms. In this study, marine fungi with conspicuous subglobose sequestrate basidioma were discovered from mangrove forests in South China. They grow on the deadwood of mangroves in the intertidal zone, periodically submerging into seawater due to the tide. Some marine animals were observed to nest in their basidiomata or consume them as food. The pileus-gleba-inner veil complex (PGI) of the basidioma was observed to be detached from the stipe and transferred into seawater by external forces, and drifting on sea to spread spores after maturity. The detachment mechanism of their PGIs was revealed through detailed microscopic observations. The contrast culturing experiment using freshwater and seawater potato dextrose agar media showed they have probably obligately adapted to the marine environment. Based on morphological and molecular phylogenetic evidence, two new species of Candolleomyces (Basidiomycota, Agaricales), namely C. brunneovagabundus and C. albovagabundus, were described. They are similar and close to each other, but can be distinguished by the size and color of the basidioma, and the size of the basidiospores.