Fig 1.
Cultivation of mature C. sinensis specimens in paper cups and collection of ascospores.
Mature C. sinensis specimens were cultivated in our Xining laboratory (altitude of 2,200 m) (Fig 1A). The fully ejected ascospores were collected using the double layers of autoclaved weighing papers (Fig 1B). Numerous semiejected ascospores adhere to the outer surface of an ascus (Fig 1C, after removing the upper layer of autoclaved weighing papers for collection of the fully ejected ascospores) during the massive ejection of ascospores. The stromal fertile portion (SFP) densely covered with numerous ascocarps is labeled with “]”.
Fig 2.
Alignment of the ITS sequences of GC- and AT-biased genotypes of O. sinensis with multiple transition point mutations.
GT represents genotype. Genotypes #1–3 are GC-biased O. sinensis genotypes, and Genotypes #4–6 and #15–17 are AT-biased O. sinensis genotypes. The sequence segments shown in blue correspond to the primers designed based on the sequences of GC-biased genotypes, and those in red correspond to primers designed based on the sequences of AT-biased genotypes. The underlined “GAATTC” site shown in green is the enzymatic site of the EcoRI endonuclease, and it is present in the GC-biased sequences at nucleotides 294–299 in Genotype #1 but absent in the AT-biased sequences due to a single-base mutation (GAATTT). “(RC)” denotes the reverse complement sequence of the primers; “-” represents identical bases; and spaces indicate unmatched sequence gaps.
Table 1.
ITS5/ITS4 universal primers and genotype- and species-specific primers used for the PCR amplification and sequencing of ITS segments.
Fig 3.
Microscopy images of fully ejected ascospores of C. sinensis without staining (upper panel; 40x) or after staining with 0.01% calmodulin for visualization of the septa of multicellular ascospores (lower panel; 400x).
Fig 4.
Microscopy images of the SFP, ascocarps and ascospores of C. sinensis.
Fig 4A is a confocal image of a transverse section of the SFP (bar, 500 μm). Fig 4B is an optical microscopic image (10x) of several C. sinensis ascocarps stained with hematoxylin-eosin. Fig 4C is a close-up optical image (40x) of an ascocarp stained with hematoxylin-eosin. Fig 4D and 4E are close-up confocal images showing ascospores gathering toward the opening of the perithecium (Fig 4D; bar, 50 μm) and a semiejected ascospore hanging out of the opening of the perithecium (Fig 4E; bar, 20 μm). Arrows in Fig 4B and 4E indicate semiejected ascospores.
Fig 5.
Agarose gel electrophoresis of the PCR amplicons obtained from genomic DNA of the fully and semiejected ascospores of C. sinensis using the Samsoniella hepiali-specific Php4/Php6 primers.
Lane M shows the molecular weight standard. Lanes 1 and 3 display the amplicon moieties amplified from the genomic DNA of the fully ejected ascospores. Lanes 2 and 4 show the amplicons amplified from the genomic DNA of the semiejected ascospores.
Table 2.
Differential occurrence of multiple genotypes of O. sinensis, S. hepiali and AB067719-type fungus in the compartments of natural C. sinensis.
Fig 6.
Schematic representation of the ITS segment sequences of the parental fungi (H. sinensis and the AB067719-type fungus) and O. sinensis offspring Genotypes #13–14.
The green bars indicate the ITS1 segment; the pink bars refer to the 5.8S gene; the blue bars represent the ITS2 segment. AB067719 [32] and KT339197 discovered in the current study represent the AB067719-type Group-E fungus and are shown with lighter bars. AB067721 [32] and KT339196 discovered in the current study represent Genotype #1 H. sinensis (Group-A by [66]) and are shown with darker bars. Alignment of the AB067719 and AB067721 sequences is shown between the lighter bars for AB067719 and the darker bars for AB067721. KT339190 and KT339178 represent O. sinensis offspring Genotypes #13 and #14, respectively, showing large DNA segment reciprocal substitutions and genetic material recombination between the genomes of the 2 parental fungi, H. sinensis and the AB067719-type fungus.
Table 3.
Sequence similarities of the ITS1, 5.8S and ITS2 segments of Genotype #1 and AB067719-type sequences compared with the multiple genotypes of O. sinensis.
Fig 7.
Bayesian phylogenetic analysis of multiple genotypes of O. sinensis.
Five ITS sequences of the whole genomes (ANOV01021709, LKHE01000582, LWBQ01000008, JAAVMX010000002 and JAAVMX010000019) of H. sinensis strains (Co18, 1229, ZJB12195 and IOZ07) and 71 ITS sequences of 17 genotypes of O. sinensis were analyzed. The Bayesian majority-rule consensus tree was inferred using MrBayes v3.2.7a software (Markov chain Monte Carlo [MCMC] algorithm) [75]. GC-biased Genotypes #1–3 and #7–14 of O. sinensis are indicated in blue alongside the tree, and the AT-biased Genotypes #4–6 and #15–17 of O. sinensis are indicated in red alongside the tree. The AB067719-type Group-E sequences (5 sequences) are indicated in purple alongside the tree as an outgroup control.
Fig 8.
Dynamic alterations of the abundance of the amplicons of Genotypes #1 and #2 of O. sinensis in the stromata of C. sinensis during maturation (modified from Fig 6 of [28]).
Immature C. sinensis had very short stroma of 1.5 cm. Maturing C. sinensis had stroma of 4.0 cm without an expanded fertile portion close to the stroma tip. Mature C. sinensis had long stroma of 7.0 cm and showed the formation of an expanded fertile portion close to the stroma tip, which was densely covered with ascocarps.