Cytological studies reveal high variation in ascospore number and shape and conidia produced directly from ascospores in Morchella galilaea.

Spores are important as dispersal and survival propagules in fungi. In this study we investigated the variation in number, shape, size and germination mode of ascospores in Morchella galilaea, the only species of the genus Morchella known to fruit in the autumn. Based on the observation of five samples, we first discovered significant variation in the shape and size of ascospores in Morchella. One to sixteen ascospores were found in the asci. Ascospore size correlated negatively with ascospore number, but positively with ascus size, and ascus size was positively correlated with ascospore number. We noted that ascospores, both from fresh collections and dried specimens, germinated terminally or laterally either by extended germ tubes, or via the production of conidia that were formed directly from ascospores at one, two or multiple sites. The direct formation of conidia from ascospores takes place within asci or after ascospores are discharged. Using laser confocal microscopy, we recorded the number of nuclei in ascospores and in conidia produced from ascospores. In most ascospores of M. galilaea, several nuclei were observed, as is typical of species of Morchella. However, nuclear number varied from zero to around 20 in this species, and larger ascospores harbored more nuclei. One to six nuclei were present in the conidia. Nuclear migration from ascospores to conidia was observed. Conidia forming directly from ascospores has been observed in few species of Pezizomycetes; this is the first report of the phenomenon in Morchella species. Morphological and molecular data show that conidial formation from ascospores is not found in all the specimens of this species and, hence, is not an informative taxonomic character in M. galilaea. Our data suggest that conidia produced from ascospores and successive mitosis within the ascus may contribute to asci with more than eight spores. The absence of mitosis and/or nuclear degeneration, as well as cytokinesis defect, likely results in asci with fewer than eight ascospores. This study provides new insights into the poorly understood life cycle of Morchella species and more broadly improves knowledge of conidia formation and reproductive strategies in Pezizomycetes.

Mating Systems in True Morels (Morchella).

True morels (Morchella spp., Morchellaceae, Ascomycota) are widely regarded as a highly prized delicacy and are of great economic and scientific value. Recently, the rapid development of cultivation technology and expansion of areas for artificial morel cultivation have propelled morel research into a hot topic. Many studies have been conducted in various aspects of morel biology, but despite this, cultivation sites still frequently report failure to fruit or only low production of fruiting bodies. Key problems include the gap between cultivation practices and basic knowledge of morel biology. In this review, in an effort to highlight the mating systems, evolution, and life cycle of morels, we summarize the current state of knowledge of morel sexual reproduction, the structure and evolution of mating-type genes, the sexual process itself, and the influence of mating-type genes on the asexual stages and conidium production. Understanding of these processes is critical for improving technology for the cultivation of morels and for scaling up their commercial production. Morel species may well be good candidates as model species for improving sexual development research in ascomycetes in the future.

Successful induction and recognition of conidiation, conidial germination and chlamydospore formation in pure culture of Morchella.

Morels, fungi from the genus Morchella, are popular edible mushrooms. However, little knowledge of their asexual reproduction and inaccessible pure mitospores hamper illumination of their life cycle. Herein, we successfully induced conidiation, conidial germination and chlamydospore formation in pure culture of Morchella sextelata. Conidiation proceeded via four morphologically distinct stages: development of the conidiophore stalk, stalk branching, phialide differentiation, and conidium production. Terminal and intercalary chlamydospores were formed on conidial hyphae. The development of conidiophores occurred earlier, with more conidia produced, in cross-mating cultures than in single-spore cultures. Mature conidia were spherical and 2.5-8 µm in diameter, with a vast majority (nearly 99%) 2.5-5 µm in diameter. Each conidium contained one to three nuclei (80.2% conidia contained one nucleus, 19.1% contained two nuclei, and 0.7% contained three nuclei). The conidial nucleus diameter was 1-2 µm. The nuclear mitosis in detached conidia that was observed may benefit their colony initiation. Additionally, morel conidia formed conidial anastomosis tubes. Conidia (mitospores) likely not only function as spermatia, but also as reproductive propagules in Morchella. Further research is imperative to elucidate the relationship between the conidia and chlamydospores, and their unique function in the morel life cycle.

Heterothallism and potential hybridization events inferred for twenty-two yellow morel species.

Mating-type genes are central to sexual reproduction in ascomycete fungi and result in the establishment of reproductive barriers. Together with hybridization, they both play important roles in the evolution of fungi. Recently, potential hybridization events and MAT genes were separately found in the Elata Clade of Morchella. Herein, we characterized the MAT1-1-1 and MAT1-2-1 genes of twenty-two species in the Esculenta Clade, another main group in the genus Morchella, and proved heterothallism to be the predominant mating strategy among the twenty-two species tested. Ascospores of these species were multi-nuclear and had many mitochondrial nucleoids. The number of ascospore nuclei might be positively related with the species distribution range. Phylogenetic analyses of MAT1-1-1, MAT1-2-1, intergenic spacer (IGS), and partial histone acetyltransferase ELP3 (F1) were performed and compared with the species phylogeny framework derived from the ribosomal internal transcribed spacer region (ITS) and translation elongation factor 1-alpha (EF1-a) to evaluate their species delimitation ability and investigate potential hybridization events. Conflicting topologies among these genes genealogies and the species phylogeny were revealed and hybridization events were detected between several species. Different evolutionary patterns were suggested for MAT genes between the Esculenta and the Elata Clades. Complex evolutionary trajectories of MAT1-1-1, MAT1-2-1, F1 and IGS in the Esculenta Clade were highlighted. These findings contribute to a better understanding of the importance of hybridization and gene transfer in Morchella and especially for the appearance of reproductive modes during its evolutionary process.

Six new species and two new records of Morchella in China using phylogenetic and morphological analyses.

In this paper, species of the genus Morchella are investigated in China. Based on morphological characteristics and molecular phylogenetic analyses of the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and the combined data set ITS + nuclear large subunit rDNA (28S) + the translation elongation factor 1-α (TEF1) gene + RNA polymerase II first largest subunit (RPB1) + RNA polymerase II second largest subunit (RPB2), six new phylogenetic species are illustrated and described: M. clivicola, M. confusa, M. odonnellii, M. owneri, M. yangii, and M. yishuica. Furthermore, two new record species, M. dunensis and M. palazonii, which were only known in Europe, are now reported for the first time from Asia. New species of morels will provide additional information on species diversity and genetic resource candidates for improving the cultivation of this economically important fungus.

Hybridization, characterization and transferability of SSRs in the genus Morchella.

Recently, Morchella importuna, M. sextelata, M. eximia, M. exuberans, Mel-13, and Mel-21 have been successfully cultivated in China and planting areas rapidly expanded because of their economic importance. Effective molecular markers are urgently needed for accurately identifying morel cultivars. Microsatellites are widely utilized for strain authentication in many fungal groups. To our knowledge, for the first time we characterized the distribution of microsatellites (simple sequence repeats, SSRs) in the M. importuna genome with 12902 SSRs and reported the first set of SSRs developed for Morchella species. Mono-nucleotides (66.2 %) were the most frequent motifs, followed by tri- (15.4 %), di- (12.1 %), tetra- (3.7 %), penta- (1.3 %) and hexa-nucleotides (1.3 %). We tested the cross-species amplification of 180 SSRs on 24 samples from the six species and high cross-species transferability of SSRs (87.7 %) was found. Among twenty-two microsatellites selected for genetic diversity analysis on 127 samples from the six species, fifteen to twenty polymorphic loci were identified in M. importuna, M. sextelata, M. eximia, M. exuberans, Mel-13 and Mel-21. Interspecific hybridization events were detected among morel species, indicating the potential application of morel crossbreeding. Ninety-one cultivated samples were characterized as new cultivars with different genotypes, but cultivar names used for these by farmers was confusing, with misnaming, synonyms and homonyms. Our results are not only helpful for cultivar identification and morel breeding programs in China, but also provide molecular tools for genetic studies in morels.

Mixed-reproductive strategies, competitive mating-type distribution and life cycle of fourteen black morel species.

Morchella species are well known world-round as popular and prized edible fungi due to their unique culinary flavor. Recently, several species have been successfully cultivated in China. However, their reproductive modes are still unknown, and their basic biology needs to be elucidated. Here, we use the morel genome information to investigate mating systems and life cycles of fourteen black morel species. Mating type-specific primers were developed to screen and genotype ascospores, hymenia and stipes from 223 ascocarps of the 14 species from Asia and Europe. Our data indicated that they are all heterothallic and their life cycles are predominantly haploid, but sterile haploid fruiting also exists. Ascospores in all species are mostly haploid, homokaryotic, and multinuclear, whereas aborted ascospores without any nuclei were also detected. Interestingly, we monitored divergent spatial distribution of both mating types in natural morel populations and cultivated sites, where the fertile tissue of fruiting bodies usually harbored both mating types, whereas sterile tissue of wild morels constantly had one MAT allele, while the sterile tissue of cultivated strains always exhibited both MAT alleles. Furthermore, MAT1-1-1 was detected significantly more commonly than MAT1-2-1 in natural populations, which strongly suggested a competitive advantage for MAT1-1 strains.

High inbreeding, limited recombination and divergent evolutionary patterns between two sympatric morel species in China.

As highly prized, popular mushrooms, morels are widely distributed in the northern hemisphere, with China as a modern centre of speciation and diversity. Overharvesting of morels has caused concern over how to effectively preserve their biological and genetic diversity. However, little is known about their population biology and life cycle. In this study, we selected two sympatric phylogenetic species, Mel-13 (124 collections from 11 geographical locations) and Morchella eohespera (156 collections from 14 geographical locations), using fragments of 4 DNA sequences, to analyse their genetic structure. Our results indicated significant differentiation among geographic locations in both species, whereas no obvious correlation between genetic and geographic distance was identified in either species. M. eohespera exhibited a predominantly clonal population structure with limited recombination detected in only 1 of the 14 geographic locations. In contrast, relatively frequent recombination was identified in 6 of the 11 geographic locations of Mel-13. Our analysis indicated that the sympatric species Mel-13 and M. eohespera might have divergent evolutionary patterns, with the former showing signatures of recent population expansion and the latter being relatively stable. Interestingly, we found no heterozygosity but strong evidence for genealogical incongruence, indicating a high level of inbreeding and hybridisation among morel species.

A review on research advances, issues, and perspectives of morels.

Morels, a group of the world's most prized edible and medicinal mushrooms, are of very important economic and scientific value. Here, we review recent research progress in the genus Morchella, and focus on its taxonomy, species diversity and distribution, ecological diversity, phylogeny and biogeography, artificial cultivation, and genome. We also discuss the potential issues remaining in the current research and suggest some future directions for study.

How well do ITS rDNA sequences differentiate species of true morels (Morchella)?

Arguably more mycophiles hunt true morels (Morchella) during their brief fruiting season each spring in the northern hemisphere than any other wild edible fungus. Concerns about overharvesting by individual collectors and commercial enterprises make it essential that science-based management practices and conservation policies are developed to ensure the sustainability of commercial harvests and to protect and preserve morel species diversity. Therefore, the primary objectives of the present study were to: (i) investigate the utility of the ITS rDNA locus for identifying Morchella species, using phylogenetic species previously inferred from multilocus DNA sequence data as a reference; and (ii) clarify insufficiently identified sequences and determine whether the named sequences in GenBank were identified correctly. To this end, we generated 553 Morchella ITS rDNA sequences and downloaded 312 additional ones generated by other researchers from GenBank using emerencia and analyzed them phylogenetically. Three major findings emerged: (i) ITS rDNA sequences were useful in identifying 48/62 (77.4%) of the known phylospecies; however, they failed to identify 12 of the 22 species within the species-rich Elata Subclade and two closely related species in the Esculenta Clade; (ii) at least 66% of the named Morchella sequences in GenBank are misidentified; and (iii) ITS rDNA sequences of up to six putatively novel Morchella species were represented in GenBank. Recognizing the need for a dedicated Web-accessible reference database to facilitate the rapid identification of known and novel species, we constructed Morchella MLST (http://www.cbs.knaw.nl/morchella/), which can be queried with ITS rDNA sequences and those of the four other genes used in our prior multilocus molecular systematic studies of this charismatic genus.

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.