Zhu L. Yang

DNA Sequence Analyses Reveal Abundant Diversity, Endemism and Evidence for Asian Origin of the Porcini Mushrooms

The wild gourmet mushroom Boletus edulis and its close allies are of significant ecological and economic importance. They are found throughout the Northern Hemisphere, but despite their ubiquity there are still many unresolved issues with regard to the taxonomy, systematics and biogeography of this group of mushrooms. Most phylogenetic studies of Boletus so far have characterized samples from North America and Europe and little information is available on samples from other areas, including the ecologically and geographically diverse regions of China. Here we analyzed DNA sequence variation in three gene markers from samples of these mushrooms from across China and compared our findings with those from other representative regions. Our results revealed fifteen novel phylogenetic species (about one-third of the known species) and a newly identified lineage represented by Boletus sp. HKAS71346 from tropical Asia. The phylogenetic analyses support eastern Asia as the center of diversity for the porcini sensu stricto clade. Within this clade, B. edulis is the only known holarctic species. The majority of the other phylogenetic species are geographically restricted in their distributions. Furthermore, molecular dating and geological evidence suggest that this group of mushrooms originated during the Eocene in eastern Asia, followed by dispersal to and subsequent speciation in other parts of Asia, Europe, and the Americas from the middle Miocene through the early Pliocene. In contrast to the ancient dispersal of porcini in the strict sense in the Northern Hemisphere, the occurrence of B. reticulatus and B. edulis sensu lato in the Southern Hemisphere was probably due to recent human-mediated introductions.

Molecular phylogenetic analyses redefine seven major clades and reveal 22 new generic clades in the fungal family Boletaceae

Mushrooms in the basidiomycete family Boletaceae are ecologically and economically very important. However, due to the morphological complexity and the limited phylogenetic information on the various species and genera of this fungal family, our understanding of its systematics and evolution remains rudimentary. In this study, DNA sequences of four genes (nrLSU, tef1-α, rpb1, and rpb2) were newly obtained from ca. 200 representative specimens of Boletaceae. Our phylogenetic analyses revealed seven major clades at the subfamily level, namely Austroboletoideae, Boletoideae, Chalciporoideae, Leccinoideae, Xerocomoideae, Zangioideae, and the Pulveroboletus Group. In addition, 59 genus-level clades were identified, of which 22 were uncovered for the first time. These 22 clades were mainly placed in Boletoideae and the Pulveroboletus Group. The results further indicated that the characters frequently used in the morphology-based taxonomy of Boletaceae, such as basidiospore ornamentation, the form of the basidioma, and the stuffed pores each had multiple origins within the family, suggesting that the use of such features for high-level classification of Boletaceae should be de-emphasized and combined with other characters.

Multigene molecular phylogenetics reveals true morels (Morchella) are especially species-rich in China

The phylogenetic diversity of true morels (Morchella) in China was estimated by initially analyzing nuclear ribosomal internal transcribed spacer (ITS) rDNA sequences from 361 specimens collected in 21 provinces during the 2003-2011 growing seasons, together with six collections obtained on loan from three Chinese herbaria. Based on the results of this preliminary screen, 40 Esculenta Clade (yellow morels) and 30 Elata Clade (black morels) were chosen to represent the full range of phylogenetic diversity sampled. To investigate their species limits, we generated DNA sequences from portions of three protein-coding genes (RPB1, RPB2 and EF-1α) and domains D1 and D2 of the nuclear large subunit (LSU) rDNA for all 70 collections. To fully assess evolutionary relationships, previously published multilocus DNA sequence data representing all known Morchella species was included in this study. Phylogenetic analyses employing maximum parsimony and maximum likelihood frameworks resolved 30 species in China compared with 22 in Europe and 19 within North America. Eleven novel phylogenetically distinct species were discovered in China, including two species within the Elata Clade and nine within the Esculenta Clade. Of the 30 species in China, 20 appear to be endemic, nine were also represented in Europe, and four putatively fire-adapted species have disjunct distributions in China, Europe and western North America. Although the diversification time estimates place the Esculenta Clade in China as early as the late Cretaceous and the Elata Clade by the early Oligocene, 27 of the 30 species evolved between the middle Miocene 12Mya and present.

Zangia, a new genus of Boletaceae supported by molecular and morphological evidence

A new distinct genus of Boletales, Zangia, with phenotypic similarities to the genus Tylopilus, is proposed based on molecular and morphological data. The monophyly of Zangia was highly supported using two nuclear and three mitochondrial genes based on Maximum Parsimony, Maximum Likelihood and Bayesian analyses. Morphologically, Zangia is distinguished from other boletoid lineages by its combination of rugose pileus, pinkish to pink hymenophore, pink to pinkish brown spore deposit, pink scabrous squamules on the stipe, chrome yellow to golden yellow stipe base, chrome yellow to golden yellow mycelia on the base of the stipe, ixohyphoepithelium pileipellis, glabrous spores and bluish colour changes in the stipe in some species. Geographically, Zangia is currently only known from southern, southeastern and southwestern China under forests dominated by Fagaceae mixed with Pinaceae. Six species, including 4 new ones and 2 new combinations, are fully documented with taxonomic descriptions and illustrations. A key to the species in Zangia is provided. It is suggested that some of the species might have started diverging from each other relatively recently with the uplifts of the eastern Himalayas and Hengduan Mountains, and both the mycorrhizal host specificity or preference and geographic separation could contribute to their ongoing divergence.

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

BackgroundLethal 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.ResultLethal 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.ConclusionThe 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.

Recombination and genetic differentiation among natural populations of the ectomycorrhizal mushroom Tricholoma matsutake from southwestern China

Effective conservation and utilization strategies for natural biological resources require a clear understanding of the natural populations of the target organisms. Tricholoma matsutake is an ectomycorrhizal mushroom that forms symbiotic associations with plants and plays an important ecological role in natural forest ecosystems in many parts of the world. It is also an economically very important gourmet mushroom. Because no artificial cultivation is available, natural populations of this species are under increasing threats, primarily from habitat disturbance and destruction. Despite its economical and ecological importance, little is known about its genetics and population biology. Here, using 14 polymerase chain reaction–restriction fragment length polymorphism markers, we analysed 154 strains from 17 geographical locations in southwestern China, a region where over 25% of the global T. matsutake harvest comes from. Our results revealed abundant genetic variation within individual populations. The analyses of gene and genotype frequencies within populations indicated that most loci did not deviate from Hardy–Weinberg equilibrium in most populations and that alleles among loci were in linkage equilibrium in the majority of the local populations. These results are consistent with the hypothesis that sexual reproduction and recombination play an important role in natural populations of this species. Our analyses indicated low but significant genetic differentiation among the geographical populations, with a significant positive correlation between genetic distance and geographical distance. We discuss the implications of our results to the ecology and resource management of this species.

Lethal amanitas of East Asia characterized by morphological and molecular data

This study investigated the morphological characteristics and molecular phylogenetic relationships among lethal Amanita species occurring in East Asia. The results revealed that at least nine phylogenetic taxa exist in the region. Among them, five were identical to previously known morphological taxa including A. exitialis, A. fuliginea, A. subjunquillea, A. subjunquillea var. alba and A. virosa; three new taxa, A. fuligineoides, A. rimosa and A. pallidorosea were described and illustrated here. The remaining one was labeled “A. sp. 1” because no robust morphological evidence was found to distinguish it from A. subjunquillea var. alba. The occurrence of A. virosa in East Asia, a well-known lethally poisonous mushroom originally described from Europe, was confirmed by both ITS sequences and morphology. Another lethal species native to Europe, A. phalloides, was determined as closely related to A. subjunquillea from East Asia. Amanita oberwinklerana was treated in section Phalloideae from a morphological point of view, but appeared to be a member of section Lepidella by the analyses of sequences from both the ITS regions and the large subunit of nuclear ribosomal RNA gene. Distribution features of East Asian Phalloideae and the phylogenetic relationships of these species with their counterparts from Europe and North America were also discussed. A key to species of section Phalloideae in East Asia is furnished.

The genus Phylloporus ( Boletaceae, Boletales ) from China: morphological and multilocus DNA sequence analyses

Species of the genus Phylloporus in China were investigated based on morphology and molecular phylogenetic analysis of a three-locus (nrLSU, ITS and tef-1a) DNA sequence dataset. Twenty-one phylogenetic species were recognized among the studied collections. Seven of them are described as new: P. brunneiceps, P. imbricatus, P. maculatus, P. pachycystidiatus, P. rubeolus, P. rubrosquamosus, and P. yunnanensis. In addition, four of them correspond with the previous morphology-based taxa: P. bellus, P. luxiensis, P. parvisporus, and P. rufescens. The remaining ten phylogenetic species were not described due to the paucity of the materials. A key to the Chinese morphologically recognizable taxa was provided. A preliminary biogeographical analysis showed that (1) Pylloporus species in East Asia and Southeast Asia are mostly closely related, (2) species pairs or closely related species of Phylloporus between East Asia and North/Central America are relatively common, and (3) the biogeographic relationship of Phylloporus between East Asia and Europe was supported by only a single species pair. Unexpectedly, no taxa common either to both Europe and East Asia, or to both East Asia and North/Central America, were uncovered. Clades look to have taxa from both sides of the Pacific and Europe/Asia though.

Ectomycorrhizal fungi associated with two species of Kobresia in an alpine meadow in the eastern Himalaya.

The diversity of ectomycorrhizal fungi (EMF) on Kobresia filicina and Kobresia capillifolia in an alpine meadow in China’s southwestern mountains, one of the word’s hotspots of biodiversity, was estimated based on internal transcribed spacer rDNA sequence analysis of root tips. Seventy EMF operational taxonomical units (OTUs) were found in the two plant species. Dauciform roots with EMF were detected in species of Kobresia for the first time. OTU richness of EMF was high in Tomentella/Thelophora and Inocybe, followed by Cortinarius, Sebacina, the Cenococcum geophilum complex, and Russula. Tomentella/Thelophora and Inocybe were general and dominant mycobiont genera of the two sedges. Besides the C. geophilum complex, the ascomycete components Hymenoscyphus and Lachnum were also detected on the two plants. Alpine plants in different geographical regions share similar main genera and/or families of EMF while harboring predominantly different mycobiont species; most of the members detected by us have not been found elsewhere. Significant differences in the profile of EMF occurrences were not found between the two plant species and among the three sampling seasons in our sample size.

Corneroboletus, a new genus to accommodate the southeastern Asian Boletus indecorus

Corneroboletus was erected in the Boletaceae to accommodate Boletus indecorus originally described from southeastern Asia. The mucilaginous surface of the basidioma, the ixohyphoepithelium pileipellis and the irregularly warty to irregularly bacillate ornamentation of basidiospores distinguish this fungus from other described species in Boletaceae. Phylogenetic placement of this fungus was investigated further with molecular data including LSU rRNA and concatenated alignment of nrLSU, 5.8S rRNA and rpb2 genes, and an independent lineage among existing genera of Boletaceae was suggested by our phylogenetic results. Consequently a description, illustrations and a comparison of Corneroboletus with allied taxa are presented.