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.

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.

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.

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.